Oligomeric compounds comprising bicyclic nucleotides and uses thereof

ABSTRACT

The present invention provides oligomeric compounds. Certain such oligomeric compounds are useful for hybridizing to a complementary nucleic acid, including but not limited, to nucleic acids in a cell. In certain embodiments, hybridization results in modulation of the amount activity or expression of the target nucleic acid in a cell.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled CORE0094USC1SEQ_ST25.txt, created Jun. 5, 2018, which is 12 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Antisense compounds have been used to modulate target nucleic acids. Antisense compounds comprising a variety of chemical modifications and motifs have been reported. In certain instances, such compounds are useful as research tools, diagnostic reagents, and as therapeutic agents. In certain instances antisense compounds have been shown to modulate protein expression by binding to a target messenger RNA (mRNA) encoding the protein. In certain instances, such binding of an antisense compound to its target mRNA results in cleavage of the mRNA. Antisense compounds that modulate processing of a pre-mRNA have also been reported. Such antisense compounds alter splicing, interfere with polyadenylation or prevent formation of the 5′-cap of a pre-mRNA.

Certain antisense compounds have been described previously. See for example U.S. Pat. No. 7,399,845 and published International Patent Application No. WO 2008/049085, which are hereby incorporated by reference herein in their entirety.

SUMMARY OF THE INVENTION

In certain embodiments, the present invention provides compounds comprising oligonucleotides. In certain embodiments, such oligonucleotides comprise a gapmer region. In certain embodiments, such oligonucleotides consist of a gapmer region.

The present disclosure provides the following non-limiting numbered embodiments:

-   Embodiment 1: A compound comprising: -   a modified oligonucleotide consisting of 10 to 20 linked     nucleosides, wherein the modified oligonucleotide comprises: -   a 5′-wing consisting of 2 to 5 linked nucleosides; -   a 3′-wing consisting of 2 to 5 linked nucleosides; and -   a gap between the 5′-wing and the 3′-wing consisting of 6 to 14     linked 2′-deoxynucleosides; and -   wherein at least one of the 5′-wing and the 3′-wing comprises at     least one bicyclic nucleoside; at least one of the 5′-wing and the     3′-wing comprises at least one 2′-substituted nucleoside; and -   wherein the nucleobase sequence of the modified oligonucleotide is     complementary to the nucleobase sequence of a target nucleic acid. -   Embodiment 2: The compound of embodiment 1, wherein one of the     5′-wing or the 3′-wing comprises at least one 2′-deoxynucleoside. -   Embodiment 3: The compound of embodiments 1-2, wherein each of the     5′-wing and the 3′-wing comprises at least one 2′-deoxynucleoside. -   Embodiment 4: The compound of embodiments 1-3, wherein the 3′-wing     comprises at least one 2′-deoxynucleoside. -   Embodiment 5: The compound of embodiments 1-4, wherein the 5′-wing     comprises at least one 2′-deoxynucleoside. -   Embodiment 6: The compound of any of embodiments 1-5, wherein the     5′-wing comprises at least one bicyclic nucleoside. -   Embodiment 7: The compound of any of embodiments 1-6, wherein the     3′-wing comprises at least one bicyclic nucleoside. -   Embodiment 8: The compound of any of embodiments 1-7, wherein the     5′-wing comprises at least one 2′-substituted nucleoside. -   Embodiment 9: The compound of any of embodiments 1-8, wherein the     3′-wing comprises at least one 2′-substituted nucleoside. -   Embodiment 10: A compound comprising: -   a modified oligonucleotide consisting of 10 to 20 linked     nucleosides, wherein the modified oligonucleotide comprises: -   a 5′-wing consisting of 2 to 5 linked nucleosides; -   a 3′-wing of 2 to 5 linked nucleosides; and -   a gap between the 5′ wing and the 3′ wing consisting of 6 to 14     linked 2′-deoxynucleosides; and -   wherein at least one of the 5′-wing and the 3′-wing comprises at     least one constrained ethyl nucleoside; and at least one of the     5′-wing and the 3′-wing comprises at least one 2′-substituted     nucleoside; and -   wherein the nucleobase sequence of the modified oligonucleotide is     complementary to the nucleobase sequence of a target nucleic acid. -   Embodiment 11: The compound of embodiments 1-10, wherein and at     least one of the 5′-wing and the 3′-wing comprises at least one     2′-deoxynucleoside. -   Embodiment 12: The compound of embodiments 1-11, wherein at least     one of the 5′-wing and the 3′-wing comprises both at least one     constrained ethyl nucleoside and at least one 2′-substituted     nucleoside. -   Embodiment 13: The compound of embodiments 1-12, wherein the 5′-wing     comprises at least one constrained ethyl nucleoside. -   Embodiment 14: The compound of any of embodiments 10-13, wherein the     3′-wing comprises at least one constrained ethyl nucleoside. -   Embodiment 15: The compound of any of embodiments 10-14, wherein the     5′-wing comprises at least one 2′-substituted nucleoside. -   Embodiment 16: The compound of any of embodiments 10-15, wherein the     3′-wing comprises at least one 2′-substituted nucleoside. -   Embodiment 17: The compound of any of embodiments 1-17, wherein the     modified oligonucleotide has a sugar motif described by Formula I as     follows: -   (A)_(m)-(B)_(n)-(J)_(p)-(B)_(r)-(J)_(t)-(D)_(g)-(J)_(v)-(B)_(w)-(J)_(x)-(B)_(y)-(A)_(z)     wherein:

each A is independently a 2′-substituted nucleoside;

each B is independently a bicyclic, nucleoside;

each J is independently either a 2′-substituted nucleoside or a 2′-deoxynucleoside;

each D is a 2′-deoxynucleoside;

m is 0-4; n is 0-2; p is 0-2; r is 0-2; t is 0-2; v is 0-2; w is 0-4; x is 0-2; y is 0-2; z is 0-4; and g is 6-14;

provided that:

at least one of m, n, and r is other than 0;

at least one of w and y is other than 0;

the sum of m, n, p, r, and t is from 2 to 5; and

the sum of v, w, x, y, and z is from 2 to 5.

-   Embodiment 18: A compound comprising: -   a modified oligonucleotide consisting of 10 to 20 linked     nucleosides, wherein the modified oligonucleotide has a sugar motif     described by Formula I as follows: -   (A)_(m)-(B)_(n)-(J)_(p)-(B)_(r)-(J)_(t)-(D)_(g)-(J)_(v)-(B)_(w)-(J)_(x)-(B)_(y)-(A)_(z)     wherein:

each A is independently a 2′-substituted nucleoside;

each B is independently a bicyclic nucleoside;

each J is independently either a 2′-substituted nucleoside or a 2′-deoxynucleoside;

each D is a 2′-deoxynucleoside;

m is 0-4; n is 0-2; p is 0-2; r is 0-2; t is 0-2; v is 0-2; w is 0-4; x is 0-2; y is 0-2; z is 0-4; and g is 6-14;

provided that:

at least one of m, n, and r is other than 0;

at least one of w and y is other than 0;

the sum of m, n, p, r, and t is from 2 to 5; and

the sum of v, w, x, y, and z is from 2 to 5.

-   Embodiment 19: The compound of embodiment 17 or 18, wherein at least     one bicyclic nucleoside is a constrained ethyl nucleoside. -   Embodiment 20: The compound of embodiment 17 or 18, wherein each     bicyclic nucleoside is a constrained ethyl nucleoside. -   Embodiment 21: The compound of any of embodiments 17-19, wherein at     least one bicyclic nucleoside is an LNA nucleoside. -   Embodiment 22: The compound of embodiment 17 or 18, wherein each     bicyclic nucleoside is an LNA nucleoside. -   Embodiment 23: The compound of any of embodiments 1-22, wherein the     2′-substituent of the at least one 2′-substituted nucleoside is     selected from among: OCH₃, F, OCH₂F, OCHF₂, OCF₃, OCH₂CH₃, O(CH₂)₂F,     OCH₂CHF₂, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—SCH₃,     O(CH₂)₂—OCF₃, O(CH₂)₃—N(R₄)(R₅), O(CH₂)₂—ON(R₄)(R₅),     O(CH₂)₂—O(CH₂)₂—N(R₄)(R₅), OCH₂C(═O)—N(R₄)(R₅),     OCH₂C(═O)—N(R₆)—(CH₂)₂—N(R₄)(R₅) and     O(CH₂)₂—N(R₆)—C(═NR₇)[N(R₄)(R₅)] wherein R₄, R₅, R₆ and R₇ are each,     independently, H or C₁-C₆ alkyl. -   Embodiment 24: The compound of embodiment 23, wherein the     2′-substituent of the at least one 2′-substituted nucleoside of is     selected from among: OCH₃, F, and O(CH₂)₂—OCH₃. -   Embodiment 25: The compound of embodiment 24, wherein the     2′-substituent of the at least one 2′-substituted nucleoside is     O(CH₂)₂—OCH₃. -   Embodiment 26: The compound of any of embodiments 1-22, wherein the     2′-substituent of each 2′-substituted nucleoside is selected from     among: OCH₃, F, OCH₂F, OCHF₂, OCF₃, OCH₂CH₃, O(CH₂)₂F, OCH₂CHF₂,     OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—SCH₃, O(CH₂)₂—OCF₃,     O(CH₂)₃—N(R₄)(R₅), O(CH₂)₂—ON(R₄)(R₅), O(CH₂)₂—O(CH₂)₂—N(R₄)(R₅),     OCH₂C(═O)—N(R₄)(R₅), OCH₂C(═O)—N(R₆)—(CH₂)₂—N(R₄)(R₅) and     O(CH₂)₂—N(R₆)—C(═NR₇)[N(R₄)(R₅)] wherein R₄, R₅, R₆ and R₇ are each,     independently, H or C₁-C₆ alkyl. -   Embodiment 27: The compound of embodiment 26, wherein the     2′-substituent of each 2′-substituted nucleoside of is selected from     among: OCH₃, F, and O(CH₂)₂—OCH₃. -   Embodiment 28: The compound of embodiment 27, wherein the     2′-substituent of each 2′-substituted nucleoside is O(CH₂)₂—OCH₃. -   Embodiment 29: The compound of any of embodiments 1-28, wherein the     5′-wing does not comprise a bicyclic nucleotide. -   Embodiment 30: The compound of any of embodiments 1-29, wherein the     3′-wing does not comprise a bicyclic nucleotide. -   Embodiment 31: The compound of any of embodiments 1-30, wherein the     target nucleic acid is not a Huntingtin gene transcript. -   Embodiment 32: The compound of any of embodiments 1-31, wherein the     modified oligonucleotide has a base sequence other than:

(SEQ ID NO: 1) GTGCTACCCAACCTTTCTG; (SEQ ID NO: 2) CACAGTGCTACCCAACCTT; (SEQ ID NO: 3) CAGTGCTACCCAACC; (SEQ ID NO: 4) ATATCACAGTGCTACCCAA; (SEQ ID NO: 5) GATGCTGACTTGGGCCATT; (SEQ ID NO: 6) GGGATGCTGACTTGG; (SEQ ID NO: 7) TGCCAAGGGATGCTGACTT; (SEQ ID NO: 8) AATTGTCATCACCAGAAAA; (SEQ ID NO: 9) TAAATTGTCATCACC; (SEQ ID NO: 10) ACAGTAGATGAGGGAGCAG; (SEQ ID NO: 11) ACACAGTAGATGAGG;  (SEQ ID NO: 12) AAGTGCACACAGTAGATGA; (SEQ ID NO: 13) AGCTGCAACCTGGCAACAA; (SEQ ID NO: 14) GCAGCTGCAACCTGG; or (SEQ ID NO: 15) GCAAGAGCAGCTGCAACCT.

-   Embodiment 33: The compound of any of embodiments 1-31, wherein the     oligonucleotide has a sugar motif other than:

E-K-K-(D)₉-K-K-E;

E-E-E-E-K-(D)₉-E-E-E-E-E;

E-K-K-K-(D)₉-K-K-K-E;

K-E-E-K-(D)₉-K-E-E-K;

K-D-D-K-(D)₉-K-D-D-K;

K-E-K-E-K-(D)₉-K-E-K-E-K;

K-D-K-D-K-(D)₉-K-D-K-D-K;

E-K-E-K-(D)₉-K-E-K-E;

E-E-E-E-E-K-(D)₈-E-E-E-E-E; or

E-K-E-K-E-(D)₉-E-K-E-K-E; wherein

K is a constrained ethyl nucleoside, E is a 2′-MOE substituted nucleoside, and D is a 2′-deoxynucleoside.

-   Embodiment 34: The compound of any of embodiments 1-30, wherein the     5′-wing consists of 2 linked nucleosides. -   Embodiment 35: The compound of any of embodiments 1-30, wherein the     5′-wing consists of 3 linked nucleosides. -   Embodiment 36: The compound of any of embodiments 1-30, wherein the     5′-wing consists of 4 linked nucleosides. -   Embodiment 37: The compound of any of embodiments 1-30, wherein the     5′-wing consists of 5 linked nucleosides. -   Embodiment 38: The compound of any of embodiments 1-34, wherein the     3′-wing consists of 2 linked nucleosides. -   Embodiment 39: The compound of any of embodiments 1-34, wherein the     3′-wing consists of 3 linked nucleosides. -   Embodiment 40: The compound of any of embodiments 1-34, wherein the     3′-wing consists of 4 linked nucleosides. -   Embodiment 41: The compound of any of embodiments 1-34, wherein the     3′-wing consists of 5 linked nucleosides. -   Embodiment 42: The compound of any of embodiments 1-38, wherein the     gap consists of 6 linked 2′-deoxynucleosides. -   Embodiment 43: The compound of any of embodiments 1-38, wherein the     gap consists of 7 linked 2′-deoxynucleosides. -   Embodiment 44: The compound of any of embodiments 1-38, wherein the     gap consists of 8 linked 2′-deoxynucleosides. -   Embodiment 45: The compound of any of embodiments 1-38, wherein the     gap consists of 9 linked 2′-deoxynucleosides. -   Embodiment 46: The compound of any of embodiments 1-38, wherein the     gap consists of 10 linked 2′-deoxynucleosides. -   Embodiment 47: The compound of any of embodiments 1-38, wherein the     gap consists of 11 linked 2′-deoxynucleosides. -   Embodiment 48: The compound of any of embodiments 1-38, wherein the     gap consists of 12 linked 2′-deoxynucleosides. -   Embodiment 49: The compound of any of embodiments 1-38, wherein the     gap consists of 13 linked 2′-deoxynucleosides. -   Embodiment 50: The compound of any of embodiments 1-38, wherein the     gap consists of 14 linked 2′-deoxynucleosides. -   Embodiment 51: The compound of any of embodiments 1-50, wherein the     oligonucleotide consists of 10 linked nucleosides. -   Embodiment 52: The compound of any of embodiments 1-50, wherein the     oligonucleotide consists of 11 linked nucleosides. -   Embodiment 53: The compound of any of embodiments 1-50, wherein the     oligonucleotide consists of 12 linked nucleosides. -   Embodiment 54: The compound of any of embodiments 1-50, wherein the     oligonucleotide consists of 13 linked nucleosides. -   Embodiment 55: The compound of any of embodiments 1-50, wherein the     oligonucleotide consists of 14 linked nucleosides. -   Embodiment 56: The compound of any of embodiments 1-50, wherein the     oligonucleotide consists of 15 linked nucleosides. -   Embodiment 57: The compound of any of embodiments 1-50, wherein the     oligonucleotide consists of 16 linked nucleosides. -   Embodiment 58: The compound of any of embodiments 1-50, wherein the     oligonucleotide consists of 17 linked nucleosides. -   Embodiment 59: The compound of any of embodiments 1-50, wherein the     oligonucleotide consists of 18 linked nucleosides. -   Embodiment 60: The compound of any of embodiments 1-50, wherein the     oligonucleotide consists of 19 linked nucleosides. -   Embodiment 61: The compound of any of embodiments 1-50, wherein the     oligonucleotide consists of 20 linked nucleosides. -   Embodiment 62: The compound of any of embodiments 1-50, wherein the     oligonucleotide consists of 21 linked nucleosides. -   Embodiment 63: The compound of any of embodiments 1-50, wherein the     oligonucleotide consists of 22 linked nucleosides. -   Embodiment 64: The compound of any of embodiments 1-30, wherein the     gapmer motif is selected from among: 2-10-2, 2-10-3, 2-10-4, 2-10-5,     3-10-2, 3-10-3, 3-10-4, 3-10-5, 4-10-2, 4-10-3, 4-10- 4, 4-10-5,     5-10-2, 5-10-3, 5-10-4, 5-10-5, 2-9-2, 2-9-3, 2-9-4, 2-9-5, 3-9-2,     3-9-3, 3-9-4, 3-9-5, 4-9-2, 4-9-3, 4-9-4, 4-9-5, 5-9-2, 5-9-3,     5-9-4, 5-9-5, 2-8-2, 2-8-3, 2-8-4, 2-8-5, 3-8-2, 3-8-3, 3-8-4,     3-8-5, 4-8-2, 4-8-3, 4-8-4, 4-8-5, 5-8-2, 5-8-3, 5-8-4, and 5-8-5. -   Embodiment 65: A compound comprising a modified oligonucleotide     having a sugar motif selected from among sugar motifs 1-278 as shown     in Table 4. -   Embodiment 66: The compound of any of embodiments 1-65, wherein the     5′-wing has a motif selected from among the 5′-wing motifs as shown     in Tables 1-3. -   Embodiment 67: The compound of any of embodiments 1-66, wherein the     3′-wing has a motif selected from among the 3′-wing motifs as shown     in Tables 4-6. -   Embodiment 68: The compound of any of embodiments 66-67, wherein     each A, each B, and each C are independently selected from among:     HNA and F-HNA. -   Embodiment 69: The compound of any of embodiments 1-68, wherein the     S′-wing comprises at least one F-HNA. -   Embodiment 70: The compound of any of embodiments 1-69, wherein the     3′-wing comprises at least one F-HNA. -   Embodiment 71: The compound of any of embodiments 1-68, wherein the     5′-wing comprises at least one modified nucleobase. -   Embodiment 72: The compound of any of embodiments 1-69, wherein the     3′-wing comprises at least one Modified nucleobase. -   Embodiment 73: The compound of embodiment 72, wherein the modified     nucleobase is 2-thio-thymidine. -   Embodiment 74: The compound of any of embodiments 1-73, wherein the     5′-wing has a motif selected from among the 5′-wing motifs as shown     in Tables 1-3 and the 3′-wing has a motif selected from among the     3′-wing motifs as shown in Tables 4-6. -   Embodiment 75: The compound of any of embodiments 1-74, wherein the     5′-wing has an ABABA motif, wherein each A is a modified nucleoside     and each B comprises a 2′-deoxynucleoside. -   Embodiment 76: The compound of embodiment 75, wherein the modified     nucleoside is a bicyclic nucleoside. -   Embodiment 77: The compound of embodiment 76, wherein the bicyclic     nucleoside is cEt. -   Embodiment 78: The compound of embodiment 76, wherein the bicyclic     nucleoside is LNA. -   Embodiment 79: The compound of any of embodiments 75-78 wherein the     3′-wing has a motif selected from'among: AA, AB, AC, BA, BB, BC, CA,     CB, and CC. -   Embodiment 80: The compound of embodiment 79, wherein the 3′-wing     has an AA motif. -   Embodiment 81: The compound of embodiment 80, wherein A is a     2′-substituted nucleoside. -   Embodiment 82: The compound of embodiment 80, wherein the     2′-substituted nucleoside is selected from among: OCH₃, F, OCH₂F,     OCHF₂, OCF₃, OCH₂CH₃, O(CH₂)₂F, OCH₂CHF₂, OCH₂CF₃, OCH₂—CH═CH₂,     O(CH₂)₂—OCH₃, O(CH₂)₂—SCH₃, O(CH₂)₂—OCF₃, O(CH₂)₃—N(R₄)(R₅),     O(CH₂)₂—ON(R₄)(R₅), O(CH₂)₂—O(CH₂)₂—N(R₄)(R₅), OCH₂C(═O)—N(R₄)(R₅),     OCH₂C(═O)—N(R₆)—(CH₂)₂—N(R₄)(R₅) and     O(CH₂)₂—N(R₆)—C(═NR₇)[N(R₄)(R₅)] wherein R₄, R₅, R₆ and R₇ are each,     independently, H or C₁-C₆ alkyl. -   Embodiment 83: The compound of embodiment 82, wherein the     2′-substituent of each 2′-substituted nucleoside of is selected from     among: OCH₃, F, and O(CH₂)₂—OCH₃. -   Embodiment 84: The compound of embodiment 83, wherein the     2′-substituent of each 2′-substituted nucleoside is O(CH₂)₂—OCH₃. -   Embodiment 85: The compound of any of embodiments 76-84 wherein the     gap between the 5′-wing and the 3′-wing consists of 6 to 11 linked     2′-deoxynucleosides. -   Embodiment 86: The compound of any of embodiments 76-84 wherein the     gap between the 5′-wing and the 3′-wing consists of 7 to 10 linked     2′-deoxynucleosides. -   Embodiment 87: The compound of any of embodiments 76-84 wherein the     gap between the 5′-wing and the 3′-wing consists of 10 linked     2′-deoxynucleosides. -   Embodiment 88: The compound of any of embodiments 75-87 having the     sugar motif: K-D-K-D-K-(D)₆-E-E. -   Embodiment 89: The compound of any of embodiments 75-87 having the     sugar motif: K-D-K-D-K-(D)₇-E-E. -   Embodiment 90: The compound of any of embodiments 75-87 having the     sugar motif: K-D-K-D-K-(D)₈-E-E. -   Embodiment 91: The compound of any of embodiments 75-87 having the     sugar motif: K-D-K-D-K-(D)₉-E-E. -   Embodiment 92: The compound of any of embodiments 75-87 having the     sugar motif: K-D-K-D-K-(D)₁₀-E-E. -   Embodiment 93: The compound of any of embodiments 75-87 having the     sugar motif: K-D-K-D-K-(D)₁₁-E-E. -   Embodiment 94: The compound of any of embodiments 75-87 having the     sugar motif: K-D-K-D-K-(D)₁₂-E-E. -   Embodiment 95: The compound of any of embodiments 75-87 having the     sugar motif: K-D-K-D-K-(D)₁₃-E-E. -   Embodiment 96: The compound of any of embodiments 75-87 having the     sugar motif: K-D-K-D-K-(D)₁₄-E-E. -   Embodiment 97: The compound of any of embodiments 75-87 having the     sugar motif: K-D-K-D-K-(D)₁₅-E-E. -   Embodiment 98: The compound of any of embodiments 1-97, wherein the     5′-wing has a BDBDB motif, wherein each B is a bicyclic nucleoside     and each D comprises a 2′-deoxynucleoside. -   Embodiment 99: The compound of any of embodiments 1-97, wherein the     5′-wing has a BDBDB-(D)₆₋₁₅-AA motif, wherein each B is a bicyclic     nucleoside and each D comprises a 2′-deoxynucleoside. -   Embodiment 100: The compound of any of embodiments 98-99, wherein B     is selected from among: BNA, LNA, α-L-LNA, ENA and 2′-thio LNA. -   Embodiment 101: The compound of embodiment 100, wherein B comprises     BNA. -   Embodiment 102: The compound of embodiment 100, wherein B comprises     LNA. -   Embodiment 103: The compound of embodiment 100, wherein B comprises     α-L-LNA. -   Embodiment 104: The compound of embodiment 100, wherein B comprises     ENA. -   Embodiment 105: The compound of embodiment 100, wherein B comprises     2′-thio LNA. -   Embodiment 106: The compound of any of embodiments 100 to 105,     wherein A comprises a 2′substituted nucleoside. -   Embodiment 107: The compound of cliam 106, wherein the 2′     substituted nucleoside comprises MOE. -   Embodiment 108: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-B-B-(D)₈-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 109: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-B-B-(D)₉-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 110: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-B-B-(D)₁₀-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 111: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-A-B-(D)₈-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 112: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-A-B-(D)₉-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 113: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-A-B-(D)₁₀-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 114: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-D-B-(D)₈-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 115: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-D-B-(D)₉-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 116: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-D-B-(D)₁₀-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 117: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-D-A-(D)₈-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 118: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-D-A-(D)₉-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 119: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-D-A-(D)₁₀-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 120: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-A-A-(D)₈-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 121: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-A-A-(D)₉-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 122: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-A-A-(D)₁₀-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 123: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-B-B-(D)₈-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 124: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-B-B-(D)₉-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 125: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-B-B-(D)₁₀-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 126: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-A-A-(D)₈-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 127: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-A-A-(D)₉-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 128: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-A-A-(D)₁₀-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 129: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-B-B-(D)₈-B-B-B, wherein each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 130: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-B-B-(D)₉-B-B-B, wherein each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 131: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-B-B-(D)₁₀-B-B-B, wherein each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 132: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-A-A-(D)₈-B-B-B, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 133: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-A-A-(D)₉-B-B-B, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 134: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-A-A-(D)₁₀-B-B-B, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 135: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-D-D-B-(D)₈-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 136: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-D-D-B-(D)₉-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 137: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-D-D-B-(D)₁₀-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 138: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-D-D-A-(D)₈-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 139: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-D-D-A-(D)₉-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 140: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-D-D-A-(D)₁₀-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 141: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-A-A-B-(D)₈-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 142: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-A-A-B-(D)₉-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 143: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-A-A-B-(D)₁₀-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 144: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-A-A-A-(D)₈-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 145: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-A-A-A-(D)₉-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 146: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-A-A-A-(D)₁₀-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 147: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-A-A-A-(D)₈-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 148: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-A-A-A-(D)₉-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 149: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-A-A-A-(D)₁₀-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 150: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-A-A-A-(D)₈-B-B-B, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 151: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-A-A-A-(D)₉-B-B-B, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 152: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-A-A-A-(D)₁₀-B-B-B, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 153: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-D-D-B-(D)₈-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 154: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-D-D-B-(D)₉-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 155: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-D-D-B-(D)₁₀-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 156: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-A-A-A-(D)₈-B-B-B, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 157: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-A-A-A-(D)₉-B-B-B, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 158: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-A-A-A-(D)₁₀-B-B-B, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 159: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-B-B-B-(D)₈-B-B-B, wherein each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 160: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-B-B-B-(D)₉-B-B-B, each Bis an independently selected bicyclic     nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 161: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-B-B-B-(D)₁₀-B-B-B, wherein each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 162: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-A-A-A-A-(D)₈-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 163: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-A-A-A-A-(D)₉-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 164: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-A-A-A-A-(D)₁₀-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 165: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-A-A-A-A-(D)₈-B-B-B, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 166: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-A-A-A-A-(D)₉-B-B-B, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 167: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-A-A-A-A-(D)₁₀-B-B-B, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 168: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-D-A-D-B-(D)₈-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 169: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-D-A-D-B-(D)₉-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 170: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-D-A-D-B-(D)₁₀-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 171: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-D-B-D-A-(D)₈-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 172: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-D-B-D-A-(D)₉-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside

Embodiment 173: The compound of any of embodiments 1-2, wherein the compound comprises a modified oligonucleotide having sugar motif: A-D-B-D-A-(D)₁₀-B-B-A, wherein each A is an independently selected 2′-substituted nucleoside, each B is an independently selected bicyclic nucleoside, and each D is a 2′-deoxynucleoside

-   Embodiment 174: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-D-A-D-A-(D)₈-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 175: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-D-A-D-A-(D)₉-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 176: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-D-A-D-A-(D)₁₀-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 177: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-A-A-A-B-(D)₈-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 178: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-A-A-A-B-(D)₉-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 179: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-A-A-A-B-(D)₁₀-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 180: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-A-B-A-A-(D)₈-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 181: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-A-B-A-A-(D)₉-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 182: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     A-A-B-A-A-(D)₁₀-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 183: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-A-A-A-A-(D)₈-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 184: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-A-A-A-A-(D)₉-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 185: The compound of any of embodiments 1-2, wherein the     compound comprises a modified oligonucleotide having sugar motif:     B-A-A-A-A-(D)₁₀-B-B-A, wherein each A is an independently selected     2′-substituted nucleoside, each B is an independently selected     bicyclic nucleoside, and each D is a 2′-deoxynucleoside -   Embodiment 186: The compound of any of embodiments 89-185, wherein     at least one bicyclic nucleoside is a constrained ethyl nucleoside. -   Embodiment 187: The compound of any of embodiments 89-185, wherein     each bicyclic nucleoside is a constrained ethyl nucleoside. -   Embodiment 188: The compound of any of embodiments, 89-185, wherein     at least one bicyclic nucleoside is selected from among: BNA, LNA,     α-L-LNA, ENA and 2′-thio LNA. -   Embodiment 189: The compound of any of embodiments, 89-185, wherein     at least one bicyclic nucleoside is an LNA nucleoside. -   Embodiment 190: The compound of any of embodiments 89-185, wherein     each bicyclic nucleoside is an LNA nucleoside. -   Embodiment 191: The compound of any of embodiments 89-185, wherein     the 2′-substituent of the at least one 2′-substituted nucleoside is     selected from among: OCH₃, F, OCH₂F, OCHF₂, OCF₃, OCH₂CH₃, O(CH₂)₂F,     OCH₂CHF₂, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—SCH₃,     O(CH₂)₂—OCF₃, O(CH₂)₃—N(R₄)(R₅), O(CH₂)₂—ON(R₄)(R₅),     O(CH₂)₂—O(CH₂)₂—N(R₄)(R₅), OCH₂C(═O)—N(R₄)(R₅),     OCH₂C(═O)—N(R₆)—(CH₂)₂—N(R₄)(R₅) and     O(CH₂)₂—N(R₆)—C(═NR₇)[N(R₄)(R₅)] wherein R₄, R₅, R₆ and R₇ are each,     independently, H or C₁-C₆ alkyl. -   Embodiment 192: The compound of embodiment 191, wherein the     2′-substituent of the at least one 2′-substituted nucleoside of is     selected from among: OCH₃, F, and O(CH₂)₂—OCH₃. -   Embodiment 193: The compound of embodiment 192, wherein the     2′-substituent of the at least one 2′-substituted nucleoside is     O(CH₂)₂—OCH₃. -   Embodiment 194: The compound of any of embodiments 89-185, wherein     the 2′-substituent of each 2′-substituted nucleoside is selected     from among: OCH₃, F, OCH₂F, OCHF₂, OCF₃, OCH₂CH₃, O(CH₂)₂F,     OCH₂CHF₂, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—SCH₃,     O(CH₂)₂—OCF₃, O(CH₂)₃—N(R₄)(R₅), O(CH₂)₂—ON(R₄)(R₅),     O(CH₂)₂—O(CH₂)₂—N(R₄)(R₅), OCH₂C(═O)—N(R₄)(R₅),     OCH₂C(═O)—N(R₆)—(CH₂)₂—N(R₄)(R₅) and     O(CH₂)₂—N(R₆)—C(═NR₇)[N(R₄)(R₅)] wherein R₄, R₅, R₆ and R₇ are each,     independently, H or C₁-C₆ alkyl. -   Embodiment 195: The compound of embodiment 194, wherein the     2′-substituent of each 2′-substituted nucleoside of is selected from     among: OCH₃, F, and O(CH₂)₂—OCH₃. -   Embodiment 196: The compound of embodiment 195, wherein the     2′-substituent of each 2′-substituted nucleoside is O(CH₂)₂—OCH₃. -   Embodiment 197: The compound of any of embodiments 1-196, wherein     the oligonucleotide comprises at least one modified internucleoside     linkage. -   Embodiment 198: The compound of embodiment 197, wherein each     internucleoside linkage is a modified internucleoside linkage. -   Embodiment 199: The compound of embodiment 197 or 198, wherein the     modified internucleoside linkage is a phosphorothioate linkage. -   Embodiment 200: The compound of embodiment 197 or 198, wherein the     modified internucleoside linkage is a methylphosphonate. -   Embodiment 201: The compound of any of embodiments 1-200 comprising     a conjugate. -   Embodiment 202: The compound of any of embodiments 1-201 comprising     at least one 5-methyl cytosine nucleobase. -   Embodiment 203: The compound of any of embodiments 1-202 comprising     at least one modified nucleobase. -   Embodiment 204: The compound of any of embodiments 1-203, wherein     the compound is an antisense compound. -   Embodiment 205: The compound of embodiment 204, wherein the compound     is capable of inhibiting expression of the target nucleic acid in a     cell. -   Embodiment 206: The compound of embodiment 205, wherein the compound     is capable of inhibiting expression of the target nucleic acid in a     cell by at least 50%. -   Embodiment 207: The compound of embodiment 205, wherein the compound     is capable of inhibiting expression of the target nucleic acid in a     cell by at least 80%. -   Embodiment 208: The compound of any of embodiments 205-207, wherein     the cell is in an animal. -   Embodiment 209: The compound of embodiment 208, wherein the animal     is a human. -   Embodiment 210: The compound of any of embodiments 1 to 209, wherein     bicyclic nucleoside is selected from among: BNA, LNA, α-L-LNA, ENA     and 2′-thio LNA. -   Embodiment 211: A compound of any of embodiments 1-210, comprising     not more than 6 bicyclic nucleosides. -   Embodiment 212: A compound of any of embodiments 1-210, comprising     not more than 5 bicyclic nucleosides. -   Embodiment 213: A compound of any of embodiments 1-210, comprising     not more than 4 bicyclic nucleosides. -   Embodiment 214: A compound of any of embodiments 1-210, comprising     not more than 3 bicyclic nucleosides. -   Embodiment 215: A compound of any of embodiments 1-210, comprising     not more than 2 bicyclic nucleosides. -   Embodiment 216: A compound of any of embodiments 1-210, comprising     not more than 1 bicyclic nucleoside. -   Embodiment 217: The compound of any of embodiments 211-216, wherein     the bicyclic nucleoside comprises cEt. -   Embodiment 218: The compound of any of embodiments 211-216, wherein     the bicyclic nucleoside comprises LNA. -   Embodiment 219: A pharmaceutical composition comprising the compound     according to any of embodiments 1-218 and a pharmaceutically     acceptable diluent. -   Embodiment 220: A method of modulating expression of a target     nucleic acid in a cell comprising contacting the cell with a     compound according to any of embodiments 1-218. -   Embodiment 221: A method of modulating expression of a target     nucleic acid in an animal comprising administering to the animal the     pharmaceutical composition according to embodiment 220. -   Embodiment 222: A method of manufacturing a compound according to     any of embodiments 1-219 comprising forming chemical bonds. -   Embodiment 223: The method of embodiment 222, wherein said chemical     bonds are internucleoside linkages. -   Embodiment 224: The method embodiment 222 or 223, wherein the method     is performed under conditions suitable for the preparation of     products for administration to humans. -   Embodiment 225: A method of manufacturing the pharmaceutical     composition according to embodiment 224 comprising combining the     compound according to any of embodiments 1-219 and the     pharmaceutically acceptable diluent. -   Embodiment 226: The method embodiment 225, wherein the method is     performed under conditions suitable for the preparation of products     for administration to humans. -   Embodiment 227: A compound comprising a modified oligonucleotide     having a sugar motif selected from among sugar motifs 279-615 as     shown in Table 4. -   Embodiment 228: A compound comprising: -   a modified oligonucleotide consisting of 10 to 20 linked     nucleosides, wherein the modified oligonucleotide comprises: -   a 5′-wing consisting of 2 to 5 linked nucleosides; -   a 3′-wing consisting of 2 to 5 linked nucleosides; and -   a gap between the 5′-wing and the 3′-wing consisting of 6 to 14     linked 2′-deoxynucleosides; and -   wherein the 5′-wing has a sugar modification motif selected from     among the motifs in Table 1. -   Embodiment 229: A compound comprising: -   a modified oligonucleotide consisting of 10 to 20 linked     nucleosides, wherein the modified oligonucleotide comprises: -   a 5′-wing consisting of 2 to 5 linked nucleosides; -   a 3′-wing consisting of 2 to 5 linked nucleosides; and -   a gap between the 5′-wing and the 3′-wing consisting of 6 to 14     linked 2′-deoxynucleosides; and -   wherein the 3′-wing has a sugar modification motif selected from     among the motifs in Table 2. -   Embodiment 230: A compound comprising: -   a modified oligonucleotide consisting of 10 to 20 linked     nucleosides, wherein the modified oligonucleotide comprises: -   a 5′-wing consisting of 2 to 5 linked nucleosides; -   a 3′-wing consisting of 2 to 5 linked nucleosides; and -   a gap between the 5′-wing and the 3′-wing consisting of 6 to 14     linked 2′-deoxynucleosides; and -   wherein the 5′-wing has a sugar modification motif selected from     among the motifs in Table 1 and the 3′-wing has a sugar modification     motif selected from among the motifs in Table 2. -   Embodiment 231: A compound of any of embodiments 1-16, wherein the     modified oligonucleotide has a sugar motif described by Formula II     as follows:

(J)_(m)-(B)_(n)-(J)_(p)-(B)_(r)-(A)_(t)-(D)_(g)-(A)_(v)-(B)_(w)-(J)_(x)-(B)_(y)-(J)_(z)

wherein:

each A is independently a 2′-substituted nucleoside;

each B is independently a bicyclic nucleoside;

each J is independently either a 2′-substituted nucleoside or a 2′-deoxynucleoside;

each D is a 2′-deoxynucleoside;

m is 0-4; n is 0-2; p is 0-2; r is 0-2; t is 0-2; v is 0-2; w is 0-4; x is 0-2; y is 0-2; z is 0-4; g is 6-14;

provided that:

at least one of m, n, and r is other than 0;

at least one of w and y is other than 0;

the sum of m, n, p, r, and t is from 1 to 5; and

the sum of v, w, x, y, and z is from 1 to 5.

-   Embodiment 232: A compound comprising:

a modified oligonucleotide consisting of 10 to 20 linked nucleosides, wherein the modified oligonucleotide has a sugar motif described by Formula II as follows:

(J)_(m)-(B)_(n)-(J)_(p)-(B)_(r)-(A)_(t)-(D)_(g)-(A)_(v)-(B)_(w)-(J)_(x)-(B)_(y)-(J)_(z)

wherein:

each A is independently a 2′-substituted nucleoside;

each B is independently a bicyclic nucleoside;

each J is independently either a 2′-substituted nucleoside or a 2′-deoxynucleoside;

each D is a 2′-deoxynucleoside;

m is 0-4; n is 0-2; p is 0-2; r is 0-2; t is 0-2; v is 0-2; w is 0-4; x is 0-2; y is 0-2; z is 0-4; g is 6-14;

provided that:

at least one of m, n, and r is other than 0;

at least one of w and y is other than 0;

the sum of m, n, p, r, and t is from 1 to 5; and

the sum of v, w, x, y, and z is from 1 to 5.

-   Embodiment 233: The compound of embodiment 231 or 232, wherein at     least one bicyclic nucleoside is a constrained ethyl nucleoside. -   Embodiment 234: The compound of embodiment 233, wherein each     bicyclic nucleoside is a constrained ethyl nucleoside. -   Embodiment 235: The compound of any of embodiments 231-232, wherein     at least one bicyclic nucleoside is an LNA nucleoside. -   Embodiment 236: The compound of embodiments 228-232, wherein each     bicyclic nucleoside is an LNA nucleoside. -   Embodiment 237: A method of treating a disease or condition. -   Embodiment 238: Use of a compound of any of embodiments 1 to 237 for     the preparation of a medicament for the treatment of a disease or     condition.

In certain embodiments, including but not limited to any of the above numbered embodiments, compounds including oligonucleotides described herein are capable of modulating expression of a target mRNA. In certain embodiments, the target mRNA is associated with a disease or disorder, or encodes a protein that is associated with a disease or disorder. In certain embodiments, the compounds or oligonucleotides provided herein modulate the expression of function of such mRNA to alleviate one or more symptom of the disease or disorder.

In certain embodiments, compounds including oligonucleotides describe herein are useful in vitro. In certain embodiments such compounds are used in diagnostics and/or for target validation experiments.

DETAILED DESCRIPTION OF THE INVENTION

Unless specific definitions are provided, the nomenclature used in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis, and chemical analysis. Certain such techniques and procedures may be found for example in “Carbohydrate Modifications in Antisense Research” Edited by Sangvi and Cook, American Chemical Society , Washington D.C., 1994; “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., 21^(st) edition, 2005; and “Antisense Drug Technology, Principles, Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press, Boca Raton, Fla.; and Sambrook et al., “Molecular Cloning, A laboratory Manual,” 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, which are hereby incorporated by reference for any purpose. Where permitted, all patents, applications, published applications and other publications and other data referred to throughout in the disclosure are incorporated by reference herein in their entirety.

Unless otherwise indicated, the following terms have the following meanings:

As used herein, “nucleoside” means a compound comprising a nucleobase moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (as found in DNA and RNA) and modified nucleosides. Nucleosides may be linked to a phosphate moiety.

As used herein, “chemical modification” means a chemical difference in a compound when compared to a naturally occurring counterpart. In reference to an oligonucleotide, chemical modification does not include differences only in nucleobase sequence. Chemical modifications of oligonucleotides include nucleoside modifications (including sugar moiety modifications and nucleobase modifications) and internucleoside linkage modifications.

As used herein, “furanosyl” means a structure comprising a 5-membered ring comprising four carbon atoms and one oxygen atom.

As used herein, “naturally occurring sugar moiety” means a ribofuranosyl as found in naturally occurring RNA or a deoxyribofuranosyl as found in naturally occurring DNA.

As used herein, “sugar moiety” means a naturally occurring sugar moiety or a modified sugar moiety of a nucleoside.

As used herein, “modified sugar moiety” means a substituted sugar moiety or a sugar surrogate.

As used herein, “substituted sugar moiety” means a furanosyl that is not a naturally occurring sugar moiety. Substituted sugar moieties include, but are not limited to furanosyls comprising substituents at the 2′-position, the 3′-position, the 5′-position and/or the 4′-position. Certain substituted sugar moieties are bicyclic sugar moieties.

As used herein, “2′-substituted sugar moiety” means a furanosyl comprising a substituent at the 2′-position other than H or OH. Unless otherwise indicated, a 2′-substituted sugar moiety is not a bicyclic sugar moiety (i.e., the 2′-substituent of a 2′-substituted sugar moiety does not form a bridge to another atom of the furanosyl ring.

As used herein, “MOE” means —OCH₂CH₂OCH₃.

As used herein the term “sugar surrogate” means a structure that does not comprise a furanosyl and that is capable of replacing the naturally occurring sugar moiety of a nucleoside, such that the resulting nucleoside is capable of (1) incorporation into an oligonucleotide and (2) hybridization to a complementary nucleoside. Such structures include rings comprising a different number of atoms than furanosyl (e.g., 4, 6, or 7-membered rings); replacement of the oxygen of a furanosyl with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen); or both a change in the number of atoms and a replacement of the oxygen. Such structures may also comprise substitutions corresponding to those described for substituted sugar moieties (e.g., 6-membered carbocyclic bicyclic sugar surrogates optionally comprising additional substituents). Sugar surrogates also include more complex sugar replacements (e.g., the non-ring systems of peptide nucleic acid). Sugar surrogates include without limitation morpholinos, cyclohexenyls and cyclohexitols.

As used herein, “bicyclic sugar moiety” means a modified sugar moiety comprising a 4 to 7 membered ring (including but not limited to a furanosyl) comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure. In certain embodiments, the 4 to 7 membered ring is a sugar ring. In certain embodiments the 4 to 7 membered ring is a furanosyl. In certain such embodiments, the bridge connects the 2′-carbon and the 4′-carbon of the furanosyl.

As used herein, “nucleotide” means a nucleoside further comprising a phosphate linking group. As used herein, “linked nucleosides” may or may not be linked by phosphate linkages and thus includes, but is not limited to “linked nucleotides.” As used herein, “linked nucleosides” are nucleosides that are connected in a continuous sequence (i.e. no additional nucleosides are present between those that are linked).

As used herein, “nucleobase” means a group of atoms that can be linked to a sugar moiety to create a nucleoside that is capable of incorporation into an oligonucleotide, and wherein the group of atoms is capable of bonding with a complementary naturally occurring nucleobase of another oligonucleotide or nucleic acid. Nucleobases may be naturally occurring or may be modified.

As used herein, “heterocyclic base” or “heterocyclic nucleobase” means a nucleobase comprising a heterocyclic structure.

As used herein the terms, “unmodified nucleobase” or “naturally occurring nucleobase” means the naturally occurring heterocyclic nucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) (including 5-methyl C), and uracil (U).

As used herein, “modified nucleobase” means any nucleobase that is not a naturally occurring nucleobase.

As used herein, “modified nucleoside” means a nucleoside comprising at least one chemical modification compared to naturally occurring RNA or DNA nucleosides. Modified nucleosides comprise a modified sugar moiety and/or a modified nucleobase.

As used herein, “bicyclic nucleoside” or “BNA” means a nucleoside comprising a bicyclic sugar moiety.

As used herein, “constrained ethyl nucleoside” or “cEt” means a nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH(CH₃)—O-2′bridge.

As used herein, “locked nucleic acid nucleoside” or “LNA” means a nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH₂—O-2′bridge.

As used herein, “2′-substituted nucleoside” means a nucleoside comprising a substituent at the 2′-position other than H or OH. Unless otherwise indicated, a 2′-substituted nucleoside is not a bicyclic nucleoside.

As used herein, “2′-deoxynucleoside” means a nucleoside comprising 2′-H furanosyl sugar moiety, as found in naturally occurring deoxyribonucleosides (DNA). In certain embodiments, a 2′-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (e.g., uracil).

As used herein, “oligonucleotide” means a compound comprising a plurality of linked nucleosides. In certain embodiments, an oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and/or unmodified deoxyribonucleosides (DNA) and/or one or more modified nucleosides.

As used herein “oligonucleoside” means an oligonucleotide in which none of the internucleoside linkages contains a phosphorus atom. As used herein, oligonucleotides include oligonucleosides.

As used herein, “modified oligonucleotide” means an oligonucleotide comprising at least one modified nucleoside and/or at least one modified internucleoside linkage.

As used herein “internucleoside linkage” means a covalent linkage between adjacent nucleosides in an oligonucleotide.

As used herein “naturally occurring internucleoside linkage” means a 3′ to 5′ phosphodiester linkage.

As used herein, “modified internucleoside linkage” means any internucleoside linkage other than a naturally occurring internucleoside linkage.

As used herein, “oligomeric compound” means a polymeric structure comprising two or more sub-structures. In certain embodiments, an oligomeric compound comprises an oligonucleotide. In certain embodiments, an oligomeric compound comprises one or more conjugate groups and/or terminal groups. In certain embodiments, an oligomeric compound consists of an oligonucleotide.

As used herein, “terminal group” means one or more atom attached to either, or both, the 3′ end or the 5′ end of an oligonucleotide. In certain embodiments a terminal group is a conjugate group. In certain embodiments, a terminal group comprises one or more terminal group nucleosides.

As used herein, “conjugate” means an atom or group of atoms bound to an oligonucleotide or oligomeric compound. In general, conjugate groups modify one or more properties of the compound to which they are attached, including, but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and/or clearance properties.

As used herein, “conjugate linking group” means any atom or group of atoms used to attach a conjugate to an oligonucleotide or oligomeric compound.

As used herein, “antisense compound” means a compound comprising or consisting of an oligonucleotide at least a portion of which is complementary to a target nucleic acid to which it is capable of hybridizing, resulting in at least one antisense activity.

As used herein, “antisense activity” means any detectable and/or measurable change attributable to the hybridization of an antisense compound to its target nucleic acid.

As used herein, “detecting” or “measuring” means that a test or assay for detecting or measuring is performed. Such detection and/or measuring may result in a value of zero. Thus, if a test for detection or measuring results in a finding of no activity (activity of zero), the step of detecting or measuring the activity has nevertheless been performed.

As used herein, “detectable and/or measureable activity” means a measurable activity that is not zero.

As used herein, “essentially unchanged” means little or no change in a particular parameter, particularly relative to another parameter which changes much more. In certain embodiments, a parameter is essentially unchanged when it changes less than 5%. In certain embodiments, a parameter is essentially unchanged if it changes less than two-fold while another parameter changes at least ten-fold. For example, in certain embodiments, an antisense activity is a change in the amount of a target nucleic acid. In certain such embodiments, the amount of a non-target nucleic acid is essentially unchanged if it changes much less than the target nucleic acid does, but the change need not be zero.

As used herein, “expression” means the process by which a gene ultimately results in a protein. Expression includes, but is not limited to, transcription, post-transcriptional modification (e.g., splicing, polyadenylation, addition of 5′-cap), and translation.

As used herein, “target nucleic acid” means a nucleic acid molecule to which an antisense compound hybridizes.

As used herein, “single nucleotide polymorphism” or “SNP” means a single nucleotide variation between the genomes of individuals of the same species. In some cases, a SNP may be a single nucleotide deletion or insertion.

As used herein, “mRNA” means an RNA molecule that encodes a protein.

As used herein, “pre-mRNA” means an RNA transcript that has not been fully processed into mRNA. Pre-RNA includes one or more intron.

As used herein, “object RNA” means an RNA molecule other than a target RNA, the amount, activity, splicing, and/or function of which is modulated, either directly or indirectly, by a target nucleic acid. In certain embodiments, a target nucleic acid modulates splicing of an object RNA. In certain such embodiments, an antisense compound modulates the amount or activity of the target nucleic acid, resulting in a change in the splicing of an object RNA and ultimately resulting in a change in the activity or function of the object RNA.

As used herein, “microRNA” means a naturally occurring, small, non-coding RNA that represses gene expression of at least one mRNA. In certain embodiments, a microRNA represses gene expression by binding to a target site within a 3′ untranslated region of an mRNA. In certain embodiments, a microRNA has a nucleobase sequence as set forth in miRBase, a database of published microRNA sequences found at microrna.sanger.ac.uk/sequences/. In certain embodiments, a microRNA has a nucleobase sequence as set forth in miRBase version 12.0 released September 2008, which is herein incorporated by reference in its entirety.

As used herein, “microRNA mimic” means an oligomeric compound having a sequence that is at least partially identical to that of a microRNA. In certain embodiments, a microRNA mimic comprises the microRNA seed region of a microRNA. In certain embodiments, a microRNA mimic modulates translation of more than one target nucleic acids. In certain embodiments, a microRNA mimic is double-stranded.

As used herein, “targeting” or “targeted to” means the association of an antisense compound to a particular target nucleic acid molecule or a particular region of a target nucleic acid molecule. An antisense compound targets a target nucleic acid if it is sufficiently complementary to the target nucleic acid to allow hybridization under physiological conditions.

As used herein, “nucleobase complementarity” or “complementarity” when in reference to nucleobases means a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In certain embodiments, complementary nucleobase means a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair. Nucleobases comprising certain modifications may maintain the ability to pair with a counterpart nucleobase and thus, are still capable of nucleobase complementarity.

As used herein, “non-complementary” in reference to nucleobases means a pair of nucleobases that do not form hydrogen bonds with one another.

As used herein, “complementary” in reference to oligomeric compounds (e.g., linked nucleosides, oligonucleotides, or nucleic acids) means the capacity of such oligomeric compounds or regions thereof to hybridize to another oligomeric compound or region thereof through nucleobase complementarity under stringent conditions. Complementary oligomeric compounds need not have nucleobase complementarity at each nucleoside. Rather, some mismatches are tolerated. In certain embodiments, complementary oligomeric compounds or regions are complementary at 70% of the nucleobases (70% complementary). In certain embodiments, complementary oligomeric compounds or regions are 80% complementary. In certain embodiments, complementary oligomeric compounds or regions are 90% complementary. In certain embodiments, complementary oligomeric compounds or regions are 95% complementary. In certain embodiments; complementary oligomeric compounds or regions are 100% complementary.

As used herein, “hybridization” means the pairing of complementary oligomeric compounds (e.g., an antisense compound and its target nucleic acid). While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.

As used herein, “specifically hybridizes” means the ability of an oligomeric compound to hybridize to one nucleic acid site with greater affinity than it hybridizes to another nucleic acid site. In certain embodiments, an antisense oligonucleotide specifically hybridizes to more than one target site.

As used herein, “fully complementary” in reference to an oligonucleotide or portion thereof means that each nucleobase of the oligonucleotide or portion thereof is capable of pairing with a nucleobase of a complementary nucleic acid or contiguous portion thereof. Thus, a fully complementary region comprises no mismatches or unhybridized nucleobases in either strand.

As used herein, “percent complementarity” means the percentage of nucleobases of an oligomeric compound that are complementary to an equal-length portion of a target nucleic acid. Percent complementarity is calculated by dividing the number of nucleobases of the oligomeric compound that are complementary to nucleobases at corresponding positions in the target nucleic acid by the total length of the oligomeric compound.

As used herein, “percent identity” means the number of nucleobases in a first nucleic acid that are the same type (independent of chemical modification) as nucleobases at corresponding positions in a second nucleic acid, divided by the total number of nucleobases in the first nucleic acid.

As used herein, “modulation” means a change of amount or quality of a molecule, function, or activity when compared to the amount or quality of a molecule, function, or activity prior to modulation. For example, modulation includes the change, either an increase (stimulation or induction) or a decrease (inhibition or reduction) in gene expression. As a further example, modulation of expression can include a change in splice site selection of pre-mRNA processing, resulting in a change in the absolute or relative amount of a particular splice-variant compared to the amount in the absence of modulation.

As used herein, “motif” means a pattern of chemical modifications in an oligomeric compound or a region thereof. Motifs may be defined by modifications at certain nucleosides and/or at certain linking groups of an oligomeric compound.

As used herein, “nucleoside motif” means a pattern of nucleoside modifications in an oligomeric compound or a region thereof. The linkages of such an oligomeric compound may be modified or unmodified. Unless otherwise indicated, motifs herein describing only nucleosides are intended to be nucleoside motifs. Thus, in such instances, the linkages are not limited.

As used herein, “sugar motif” means a pattern of sugar modifications in an oligomeric compound or a region thereof.

As used herein, “linkage motif” means a pattern of linkage modifications in an oligomeric compound or region thereof The nucleosides of such an oligomeric compound may be modified or unmodified. Unless otherwise indicated, motifs herein describing only linkages are intended to be linkage motifs. Thus, in such instances, the nucleosides are not limited.

As used herein, “nucleobase modification motif” means a pattern of modifications to nucleobases along an oligonucleotide. Unless otherwise indicated, a nucleobase modification motif is independent of the nucleobase sequence.

As used herein, “sequence motif” means a pattern of nucleobases arranged along an oligonucleotide or portion thereof. Unless otherwise indicated, a sequence motif is independent of chemical modifications and thus may have any combination of chemical modifications, including no chemical modifications.

As used herein, “type of modification” in reference to a nucleoside or a nucleoside of a “type” means the chemical modification of a nucleoside and includes modified and unmodified nucleosides. Accordingly, unless otherwise indicated, a “nucleoside having a modification of a first type” may be an unmodified nucleoside.

As used herein, “differently modified” mean chemical modifications or chemical substituents that are different from one another, including absence of modifications. Thus, for example, a MOE nucleoside and an unmodified DNA nucleoside are “differently modified,” even though the DNA nucleoside is unmodified. Likewise, DNA and RNA are “differently modified,” even though both are naturally-occurring unmodified nucleosides. Nucleosides that are the same but for comprising different nucleobases are not differently modified. For example, a nucleoside comprising a 2′-OMe modified sugar and an unmodified adenine nucleobase and a nucleoside comprising a 2′-OMe modified sugar and an unmodified thymine nucleobase are not differently modified.

As used herein, “the same type of modifications” refers to modifications that are the same as one another, including absence of modifications. Thus, for example, two unmodified DNA nucleoside have “the same type of modification,” even though the DNA nucleoside is unmodified. Such nucleosides having the same type modification may comprise different nucleobases.

As used herein, “separate regions” means portions of an oligonucleotide wherein the chemical modifications or the motif of chemical modifications of any neighboring portions include at least one difference to allow the separate regions to be distinguished from one another.

As used herein, “pharmaceutically acceptable carrier or diluent” means any substance suitable for use in administering to an animal. In certain embodiments, a pharmaceutically acceptable carrier or diluent is sterile saline. In certain embodiments, such sterile saline is pharmaceutical grade saline.

As used herein, “substituent” and “substituent group,” means an atom or group that replaces the atom or group of a named parent compound. For example a substituent of a modified nucleoside is any atom or group that differs from the atom or group found in a naturally occurring nucleoside (e.g., a modified 2′-substituent is any atom or group at the 2′-position of a nucleoside other than H or OH). Substituent groups can be protected or unprotected. In certain embodiments, compounds of the present invention have substituents at one or at more than one position of the parent compound. Substituents may also be further substituted with other substituent groups and may be attached directly or via a linking group such as an alkyl or hydrocarbyl group to a parent compound.

Likewise, as used herein, “substituent” in reference to a chemical functional group means an atom or group of atoms differs from the atom or a group of atoms normally present in the named functional group. In certain embodiments, a substituent replaces a hydrogen atom of the functional group (e.g., in certain embodiments, the substituent of a substituted methyl group is an atom or group other than hydrogen which replaces one of the hydrogen atoms of an unsubstituted methyl group). Unless otherwise indicated, groups amenable for use as substituents include without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (—C(O)R_(aa)), carboxyl (—C(O)O—R_(aa)), aliphatic groups, alicyclic groups, alkoxy, substituted oxy (—O—R_(aa)), aryl, aralkyl, heterocyclic radical, heteroaryl, heteroarylalkyl, amino (—N(R_(bb))(R_(cc)), imino(=NR_(bb)), amido (—C(O)N(R_(bb))(R_(cc)) or —N(R_(bb))C(O)R_(aa)), azido (—N₃), nitro (—NO₂), cyano (—CN), carbamido (—OC(O)N(R_(bb))(R_(cc)) or —N(R_(bb))C(O)OR_(aa)), ureido (—N(R_(bb))C(O)N(R_(bb))(R_(cc))), thioureido (—N(R_(bb))C(S)N(R_(bb))—(R_(cc))), guanidinyl (—N(R_(bb))C(═NR_(bb))N(R_(bb))(R_(cc))), amidinyl (—C(═NR_(bb))N(R_(bb))(R_(cc)) or —N(R_(bb))C(═NR_(bb))(R_(aa))), thiol (—SR_(bb)), sulfinyl (—S(O)R_(bb)), sulfonyl (—S(O)₂R_(bb)) and sulfonamidyl (—S(O)₂N(R_(bb))(R_(cc)) or —N(R_(bb))S—(O)₂R_(bb)). Wherein each R_(aa), R_(bb) and R_(cc) is, independently, H, an optionally linked chemical functional group or a further substituent group with a preferred list including without limitation, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic and heteroarylalkyl. Selected substituents within the compounds described herein are present to a recursive degree.

As used herein, “alkyl,” as used herein, means a saturated straight or branched hydrocarbon radical containing up to twenty four carbon atoms. Examples of alkyl groups include without limitation, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like. Alkyl groups typically include from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms (C₁-C₁₂ alkyl) with from 1 to about 6 carbon atoms being more preferred.

As used herein, “alkenyl,” means a straight or branched hydrocarbon chain radical containing up to twenty four carbon atoms and having at least one carbon-carbon double bond. Examples of alkenyl groups include without limitation, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene and the like. Alkenyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkenyl groups as used herein may optionally include one or more further substituent groups.

As used herein, “alkynyl,” means a straight or branched hydrocarbon radical containing up to twenty four carbon atoms and having at least one carbon-carbon triple bond. Examples of alkynyl groups include, without limitation, ethynyl, 1-propynyl, 1-butynyl, and the like. Alkynyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkynyl groups as used herein may optionally include one or more further substituent groups.

As used herein, “acyl,” means a radical formed by removal of a hydroxyl group from an organic acid and has the general Formula —C(O)—X where X is typically aliphatic, alicyclic or aromatic. Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphatic phosphates and the like. Acyl groups as used herein may optionally include further substituent groups.

As used herein, “alicyclic” means a cyclic ring system wherein the ring is aliphatic. The ring system can comprise one or more rings wherein at least one ring is aliphatic. Preferred alicyclics include rings having from about 5 to about 9 carbon atoms in the ring. Alicyclic as used herein may optionally include further substituent groups.

As used herein, “aliphatic” means a straight or branched hydrocarbon radical containing up to twenty four carbon atoms wherein the saturation between any two carbon atoms is a single, double or triple bond. An aliphatic group preferably contains from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms with from 1 to about 6 carbon atoms being more preferred. The straight or branched chain of an aliphatic group may be interrupted with one or more heteroatoms that include nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groups interrupted by heteroatoms include without limitation, polyalkoxys, such as polyalkylene glycols, polyamines, and polyimines. Aliphatic groups as used herein may optionally include further substituent groups.

As used herein, “alkoxy” means a radical formed between an alkyl group and an oxygen atom wherein the oxygen atom is used to attach the alkoxy group to a parent molecule. Examples of alkoxy groups include without limitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groups as used herein may optionally include further substituent groups.

As used herein, “aminoalkyl” means an amino substituted C₁-C₁₂ alkyl radical. The alkyl portion of the radical forms a covalent bond with a parent molecule. The amino group can be located at any position and the aminoalkyl group can be substituted with a further substituent group at the alkyl and/or amino portions.

As used herein, “aralkyl” and “arylalkyl” mean an aromatic group that is covalently linked to a C₁-C₁₂ alkyl radical. The alkyl radical portion of the resulting aralkyl (or arylalkyl) group forms a covalent bond with a parent molecule. Examples include without limitation, benzyl, phenethyl and the like. Aralkyl groups as used herein may optionally include further substituent groups attached to the alkyl, the aryl or both groups that form the radical group.

As used herein, “aryl” and “aromatic” mean a mono- or polycyclic carbocyclic ring system radicals having one or more aromatic rings. Examples of aryl groups include without limitation, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. Preferred aryl ring systems have from about 5 to about 20 carbon atoms in one or more rings. Aryl groups as used herein may optionally include further substituent groups.

As used herein, “halo” and “halogen,” mean an atom selected from fluorine, chlorine, bromine and iodine.

As used herein, “heteroaryl,” and “heteroaromatic,” mean a radical comprising a mono- or polycyclic aromatic ring, ring system or fused ring system wherein at least one of the rings is aromatic and includes one or more heteroatoms. Heteroaryl is also meant to include fused ring systems including systems where one or more of the fused rings contain no heteroatoms. Heteroaryl groups typically include one ring atom selected from sulfur, nitrogen or oxygen. Examples of heteroaryl groups include without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl and the like. Heteroaryl radicals can be attached to a parent molecule directly or through a linking moiety such as an aliphatic group or hetero atom. Heteroaryl groups as used herein may optionally include further substituent groups.

Oligomeric Compounds

In certain embodiments, the present invention provides oligomeric compounds. In certain embodiments, such oligomeric compounds comprise oligonucleotides optionally comprising one or more conjugate and/or terminal groups. In certain embodiments, an oligomeric compound consists of an oligonucleotide. In certain embodiments, oligonucleotides comprise one or more chemical modifications. Such chemical modifications include modifications one or more nucleoside (including modifications to the sugar moiety and/or the nucleobase) and/or modifications to one or more internucleoside linkage.

Certain Sugar Moieties

In certain embodiments, oligomeric compounds of the invention comprise one or more modified nucleosides comprising a modified sugar moiety. Such oligomeric compounds comprising one or more sugar-modified nucleosides may have desirable properties, such as enhanced nuclease stability or increased binding affinity with a target nucleic acid relative to oligomeric compounds comprising only nucleosides comprising naturally occurring sugar moieties. In certain embodiments, modified sugar moieties are substituted sugar moieties. In certain embodiments, modified sugar moieties are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of substituted sugar moieties.

In certain embodiments, modified sugar moieties are substituted sugar moieties comprising one or more non-bridging sugar substituent, including but not limited to substituents at the 2′ and/or 5′ positions. Examples of sugar substituents suitable for the 2′-position, include, but are not limited to: 2′-F, 2′-OCH₃ (“OMe” or “O-methyl”), and 2′-O(CH₂)₂OCH₃ (“MOE”). In certain embodiments, sugar substituents at the 2′ position is selected from allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, O—C₁-C₁₀ substituted alkyl; OCF₃, O(CH₂)₂SCH₃, O(CH₂)₂—O—N(R_(m))(R_(n)), and O—CH₂—C(═O)—N(R_(m))(R_(n)), where each Rm and Rn is, independently, H or substituted or unsubstituted C₁-C₁₀ alkyl. Examples of sugar substituents at the 5′-position, include, but are not limited to: 5′-methyl (R or S); 5′-vinyl, and 5′-methoxy. In certain embodiments, substituted sugars comprise more than one non-bridging sugar substituent, for example, 2′-F-5′-methyl sugar moieties (see,e.g., PCT International Application WO 2008/101157, for additional 5′,2′-bis substituted sugar moieties and nucleosides).

Nucleosides comprising 2′-substituted sugar moieties are referred to as 2′-substituted nucleosides. In certain embodiments, a 2′-substituted nucleoside comprises a 2′-substituent group selected from halo, allyl, amino, azido, SH, CN, OCN, CF₃, OCF₃, O, S, or N(R_(m))-alkyl; O, S, or N(R_(m))-alkenyl; O, S or N(R_(m))-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)) or O—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is, independently, H, an amino protecting group or substituted or unsubstituted C₁-C₁₀ alkyl. These 2′-substituent groups can be further substituted with one or more substituent groups independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO₂), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

In certain embodiments, a 2′-substituted nucleoside comprises a 2′-substituent group selected from F, NH₂, N₃, OCF₃, O—CH₃, O(CH₂)₃NH₂, CH₂—CH═CH₂, O—CH₂—CH═CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)), O(CH₂)₂O(CH₂)₂N(CH₃)₂, and N-substituted acetamide (O—CH₂—C(═O)—N(R_(m))(R_(n)) where each R_(m) and R_(n) is, independently, H, an amino protecting group or substituted or unsubstituted C₁-C₁₀ alkyl.

In certain embodiments, a 2′-substituted nucleoside comprises a sugar moiety comprising a 2′-substituent group selected from F, OCF₃, O—CH₃, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(CH₃)₂, —O(CH₂)₂O(CH₂)₂N(CH₃)₂, and O—CH₂—C(═O)—N(H)CH₃.

In certain embodiments, a 2′-substituted nucleoside comprises a sugar moiety comprising a 2′-substituent group selected from F, O—CH₃, and OCH₂CH₂OCH₃.

Certain modified sugar moieties comprise a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety comprises a bridge between the 4′ and the 2′ furanose ring atoms. Examples of such 4′ to 2′ sugar substituents, include, but are not limited to: —[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—, —C(R_(a)R_(b))—N(R)—O— or, —C(R_(a)R_(b))—O—N(R)—; 4′—CH₂-2′, 4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′, 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2′; 4′-(CH₂)₂-O-2′ (ENA); 4′-CH(CH₃)—O-2′ (cEt) and 4′-CH(CH₂OCH₃)—O-2′, and analogs thereof (see, e.g., U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-C(CH₃)(CH₃)—O-2′and analogs thereof, (see, e.g., WO2009/006478, published Jan. 8, 2009); 4′-CH₂—N(OCH₃)-2′ and analogs thereof (see, e.g., WO2008/150729, published Dec. 11, 2008); 4′-CH₂—O—N(CH₃)-2′ (see, e.g., US2004/0171570, published Sep. 2, 2004); 4′-CH₂—O—N(R)-2′, and 4′-CH₂—N(R)—O-2′-, wherein each R is, independently, H, a protecting group, or C₁-C₁₂ alkyl; 4′-CH₂—N(R)—O-2′, wherein R is H, C₁-C₁₂ alkyl, or a protecting group (see, U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4′-CH₂—C(H)(CH₃)-2′ (see, e.g., Chattopadhyaya, et al., J. Org. Chem.,2009, 74, 118-134); and 4′-CH₂—C(═CH₂)-2′ and analogs thereof (see, published PCT International Application WO 2008/154401, published on Dec. 8, 2008).

In certain embodiments, such 4′ to 2′ bridges independently comprise from 1 to 4 linked groups independently selected from —[C(R_(a))(R_(b))]_(n)—, —C(R_(a))═C(R_(b))—, —C(R_(a))═N—, —C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—, —Si(R_(a))₂—, —S(═O)_(x)—, and —N(R_(a))—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical, substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃, COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), or sulfoxyl (S(═O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl (C(═O)—H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl, or a protecting group.

Nucleosides comprising bicyclic sugar moieties are referred to as bicyclic nucleosides or BNAs. Bicyclic nucleosides include, but are not limited to, (A) α-L-Methyleneoxy (4′-CH₂—O-2′) BNA, (B) β-D-Methyleneoxy (4′-CH₂—O-2′) BNA (also referred to as locked nucleic acid or LNA), (C) Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) Aminooxy (4′-CH₂—O—N(R)-2′) BNA, (E) Oxyamino (4′-CH₂—N(R)—O-2′) BNA, (F) Methyl(methyleneoxy) (4′-CH(CH₃)—O-2′) BNA (also referred to as constrained ethyl or cEt), (G) methylene-thio (4′-CH₂—S-2′) BNA, (H) methylene-amino (4′-CH₂—N(R)-2′) BNA, (I) methyl carbocyclic (4′-CH₂—CH(CH₃)-2′) BNA, and (J) propylene carbocyclic (4′-(CH₂)₃-2′) BNA as depicted below.

wherein Bx is a nucleobase moiety and R is, independently, H, a protecting group, or C₁-C₁₂ alkyl.

Additional bicyclic sugar moieties are known in the art, for example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 129(26) 8362-8379 (Jul. 4, 2007); Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos. 7,053,207, 6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461, and 7,399,845; WO 2004/106356, WO 1994/14226, WO 2005/021570, and WO 2007/134181; U.S. Patent Publication Nos. US2004/0171570, US2007/0287831, and U52008/0039618; U.S. patent Ser. Nos. 12/129,154, 60/989,574, 61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787, and 61/099,844; and PCT International Applications Nos. PCT/US2008/064591, PCT/US2008/066154, and PCT/US2008/068922.

In certain embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration. For example, a nucleoside comprising a 4′-2′ methylene-oxy bridge, may be in the α-L configuration or in the β-D configuration. Previously, α-L-methyleneoxy (4′-CH₂—O-2′) bicyclic nucleosides have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

In certain embodiments, substituted sugar moieties comprise one or more non-bridging sugar substituent and one or more bridging sugar substituent (e.g., 5′-substituted and 4′-2′ bridged sugars). (see, PCT International Application WO 2007/134181, published on Nov. 22, 2007, wherein LNA is substituted with, for example, a 5′-methyl or a 5′-vinyl group).

In certain embodiments, modified sugar moieties are sugar surrogates. In certain such embodiments, the oxygen atom of the naturally occuring sugar is substituted, e.g., with a sulfer, carbon or nitrogen atom. In certain such embodiments, such modified sugar moiety also comprises bridging and/or non-bridging substituents as described above. For example, certain sugar surrogates comprise a 4′-sulfer atom and a substitution at the 2′-position (see,e.g., published U.S. Patent Application US2005/0130923, published on Jun. 16, 2005) and/or the 5′ position. By way of additional example, carbocyclic bicyclic nucleosides having a 4′-2′ bridge have been described (see, e.g., Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740).

In certain embodiments, sugar surrogates comprise rings having other than 5-atoms. For example, in certain embodiments, a sugar surrogate comprises a six-membered tetrahydropyran. Such tetrahydropyrans may be further modified or substituted. Nucleosides comprising such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (see Leumann, C J. Bioorg. & Med. Chem. (2002) 10:841-854), fluoro HNA (F-HNA), and those compounds having Formula VII:

wherein independently for each of said at least one tetrahydropyran nucleoside analog of Formula VII:

Bx is a nucleobase moiety;

T₃ and T4 are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of T₃ and T₄ is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugate group, or a 5′ or 3′-terminal group; q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, or substituted C₂-C₆ alkynyl; and

one of R₁ and R₂ is hydrogen and the other is selected from halogen, substituted or unsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂, and CN, wherein X is O, S or NJ₁, and each J₁, J₂, and J₃ is, independently, H or C₁-C₆ alkyl.

In certain embodiments, the modified THP nucleosides of Formula VII are provided wherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆and q₇ is other than H. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of R₁ and R₂ is F. In certain embodiments, R₁ is fluoro and R₂ is II, R₁ is methoxy and R₂ is II, and R₁ is methoxyethoxy and R₂ is II.

Many other bicyclo and tricyclo sugar surrogate ring systems are also known in the art that can be used to modify nucleosides for incorporation into antisense compounds (see, e.g., review article: Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).

Combinations of modifications are also provided without limitation, such as 2′-F-5′-methyl substituted nucleosides (see PCT International Application WO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5′,2′-bis substituted nucleosides) and replacement of the ribosyl ring oxygen atom with S and further substitution at the 2′-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5′-substitution of a bicyclic nucleic acid (see PCT International Application WO 2007/134181, published on Nov. 22, 2007 wherein a 4′-CH₂—O-2′ bicyclic nucleoside is further substituted at the 5′ position with a 5′-methyl or a 5′-vinyl group). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (see, e.g., Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).

Certain Nucleobases

In certain embodiments, nucleosides of the present invention comprise one or more unmodified nucleobases. In certain embodiments, nucleosides of the present invention comprise one or more modified nucleobases.

In certain embodiments, modified nucleobases are selected from: universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; those disclosed by Englisch et al, Angewandte Chemie, International Edition, 1991, 30, 613; and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288.

Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include without limitation, U.S. Pat. Nos. 3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985; 5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

Certain Internucleoside Linkages

In certain embodiments, the present invention provides oligomeric compounds comprising linked nucleosides. In such embodiments, nucleosides may be linked together using any internucleoside linkage. The two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters (P═O), phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates (P═S). Representative non-phosphorus containing internucleoside linking groups include, but are not limited to, methylenemethylimino (—CH₂—N(CH₃)—O—CH₂—), thiodiester (—O—C(O)—S—), thionocarbamate C(O)(NH)—S—); siloxane (—O—Si(H)₂—O—); and N,N′-dimethylhydrazine (—CH₂—N(CH₃)—N(CH₃)—). Modified linkages, compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligomeric compound. In certain embodiments, internucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers. Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing internucleoside linkages are well known to those skilled in the art.

The oligonucleotides described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), α or β such as for sugar anomers, or as (D) or (L) such as for amino acids etc. Included in the antisense compounds provided herein are all such possible isomers, as well as their racemic and optically pure forms.

Neutral internucleoside linkages include without limitation, phosphotriesters, methylphosphonates, MMI (3′-CH₂—N(CH₃)—O-5′), amide-3 (3′-CH₂—C(═O)—N(H)-5′), amide-4 (3′-CH₂—N(H)—C(═O)-5′), formacetal (3′-O—CH₂—O-5′), and thioformacetal (3′-S—CH₂—O-5′). Further neutral internucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral internucleoside linkages include nonionic linkages comprising mixed N, O, S and CH₂ component parts.

Certain Motifs

In certain embodiments, the present invention provides oligomeric compounds comprising oligonucleotides. In certain embodiments, such oligonucleotides comprise one or more chemical modification. In certain embodiments, chemically modified oligonucleotides comprise one or more modified sugars. In certain embodiments, chemically modified oligonucleotides comprise one or more modified nucleobases. In certain embodiments, chemically modified oligonucleotides comprise one or more modified internucleoside linkages. In certain embodiments, the chemically modifications (sugar modifications, nucleobase modifications, and/or linkage modifications) define a pattern or motif. In certain embodiments, the patterns of chemical modifications of sugar moieties, internucleoside linkages, and nucleobases are each independent of one another. Thus, an oligonucleotide may be described by its sugar modification motif, internucleoside linkage motif and/or nucleobase modification motif (as used herein, nucleobase modification motif describes the chemical modifications to the nucleobases independent of the sequence of nucleobases).

Certain Sugar Motifs

In certain embodiments, oligonucleotides comprise one or more type of modified sugar moieties and/or naturally occurring sugar moieties arranged along an oligonucleotide or region thereof in a defined pattern or sugar modification motif Such motifs may include any of the sugar modifications discussed herein and/or other known sugar modifications.

In certain embodiments, the oligonucleotides comprise or consist of a region having a gapmer sugar modification motif, which comprises two external regions or “wings” and an internal region or “gap.” The three regions of a gapmer motif (the 5′-wing, the gap, and the 3′-wing) form a contiguous sequence of nucleosides wherein at least some of the sugar moieties of the nucleosides of each of the wings differ from at least some of the sugar moieties of the nucleosides of the gap. Specifically, at least the sugar moieties of the nucleosides of each wing that are closest to the gap (the 3′-most nucleoside of the 5′-wing and the 5′-most nucleoside of the 3′-wing) differ from the sugar moiety of the neighboring gap nucleosides, thus defining the boundary between the wings and the gap. In certain embodiments, the sugar moieties within the gap are the same as one another. In certain embodiments, the gap includes one or more nucleoside having a sugar moiety that differs from the sugar moiety of one or more other nucleosides of the gap. In certain embodiments, the sugar modification motifs of the two wings are the same as one another (symmetric gapmer). In certain embodiments, the sugar modification motifs of the 5′-wing differs from the sugar modification motif of the 3′-wing (asymmetric gapmer).

Certain 5′-Wings

In certain embodiments, the 5′-wing of a gapmer consists of 1 to 5 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 2 to 5 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 3 to 5 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 4 or 5 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 1 to 4 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 1 to 3 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 1 or 2 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 2 to 4 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 2 or 3 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 3 or 4 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 1 nucleoside. In certain embodiments, the 5′-wing of a gapmer consists of 2 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 3linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 4 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 5 linked nucleosides.

In certain embodiments, the 5′-wing of a gapmer comprises at least one bicyclic nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least two bicyclic nucleosides. In certain embodiments, the 5′-wing of a gapmer comprises at least three bicyclic nucleosides. In certain embodiments, the 5′-wing of a gapmer comprises at least four bicyclic nucleosides. In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one LNA nucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a bicyclic nucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a constrained ethyl nucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a LNA nucleoside.

In certain embodiments, the 5′-wing of a gapmer comprises at least one non-bicyclic modified nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one 2′-substituted nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one 2′-MOE nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one 2′-OMe nucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a non-bicyclic modified nucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a 2′-substituted nucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a 2′-MOE nucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a 2′-OMe nucleoside.

In certain embodiments, the 5′-wing of a gapmer comprises at least one 2′-deoxynucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a 2′-deoxynucleoside. In a certain embodiments, the 5′-wing of a gapmer comprises at least one ribonucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a ribonucleoside.

In certain embodiments, the 5′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-substituted nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-MOE nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-OMe nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-deoxynucleoside.

In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-substituted nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-MOE nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-OMe nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-deoxynucleoside.

In certain embodiments, the 5′-wing of a gapmer comprises at least one LNA nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one LNA nucleoside and at least one 2′-substituted nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one LNA nucleoside and at least one 2′-MOE nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one LNA nucleoside and at least one 2′-OMe nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one LNA nucleoside and at least one 2′-deoxynucleoside.

In certain embodiments, the 5′-wing of a gapmer comprises at least one bicyclic nucleoside, at least one non-bicyclic modified nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one non-bicyclic modified nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one LNA nucleoside, at least one non-bicyclic modified nucleoside, and at least one 2′-deoxynucleoside.

In certain embodiments, the 5′-wing of a gapmer comprises at least one bicyclic nucleoside, at least one 2′-substituted nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one 2′-substituted nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one LNA nucleoside, at least one 2′-substituted nucleoside, and at least one 2′-deoxynucleoside.

In certain embodiments, the 5′-wing of a gapmer comprises at least one bicyclic nucleoside, at least one 2′-MOE nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one 2′-MOE nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one LNA nucleoside, at least one 2′-MOE nucleoside, and at least one 2′-deoxynucleoside.

In certain embodiments, the 5′-wing of a gapmer comprises at least one bicyclic nucleoside, at least one 2′-OMe nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one 2′-OMe nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one LNA nucleoside, at least one 2′-OMe nucleoside, and at least one 2′-deoxynucleoside.

In certain embodiments, the 5′-wing of a gapmer has a sugar motif selected from among those listed in the following non-limiting table:

TABLE 1 Certain 5′-Wing Sugar Motifs 5′- wing sugar motif # motif  1a A-B-B  2a A-A-B  3a A-D-B  4a B-D-A  5a B-A-A  6a B-B-B  7a A-A-A  8a A-D-D-B  9a B-D-D-A 10a A-A-A-B 11a B-A-A-A 12a A-A-A-A 13a B-D-D-B 14a A-A-A-A 15a B-B-B-B 16a A-A-A-A-A 17a A-D-A-D-B 18a A-D-B-D-A 19a B-D-A-D-A 20a A-A-A-A-B 21a A-A-B-A-A 22a B-A-A-A-A  1b E-B-B  2b E-E-B  3b E-D-B  4b B-D-E  5b B-E-E  6b B-B-B  7b E-E-E  8b E-D-D-B  9b B-D-D-E 10b E-E-E-B 11b B-E-E-E 12b E-E-E-E 13b B-D-D-B 14b E-E-E-E 15b B-B-B-B 16b E-E-E-E-E 17b E-D-E-D-B 18b E-D-B-D-E 19b B-D-E-D-E 20b E-E-E-E-B 21b E-E-B-E-E 22b B-E-E-E-E  1c M-B-B  2c M-M-B  3c M-D-B  4c B-D-M  5c B-M-M  6c B-B-B  7c M-M-M  8c M-D-D-B  9c B-D-D-M 10c M-M-M-B 11c B-M-M-M 12c M-M-M-M 13c B-D-D-B 14c M-M-M-M 15c B-B-B-B 16c M-M-M-M-M 17c M-D-M-D-B 18c M-D-B-D-M 19c B-D-M-D-M 20c M-M-M-M-B 21c M-M-B-M-M 22c B-M-M-M-M  1d A-L-L  2d A-A-L  3d A-D-L  4d L-D-A  5d L-A-A  6d L-L-L  7d A-A-A  8d A-D-D-L  9d L-D-D-A 10d A-A-A-L 11d L-A-A-A 12d A-A-A-A 13d L-D-D-L 14d A-A-A-A 15d L-L-L-L 16d A-A-A-A-A 17d A-D-A-D-L 18d A-D-L-D-A 19d L-D-A-D-A 20d A-A-A-A-L 21d A-A-L-A-A 22d L-A-A-A-A  1e E-L-L  2e E-E-L  3e E-D-L  4e L-D-E  5e L-E-E  6e L-L-L  7e E-E-E  8e E-D-D-L  9e L-D-D-E 10e E-E-E-L 11e L-E-E-E 12e E-E-E-E 13e L-D-D-L 14e E-E-E-E 15e L-L-L-L 16e E-E-E-E-E 17e E-D-E-D-L 18e E-D-L-D-E 19e L-D-E-D-E 20e E-E-E-E-L 21e E-E-L-E-E 22e L-E-E-E-E  1f M-L-L  2f M-M-L  3f M-D-L  4f L-D-M  5f L-M-M  6f L-L-L  7f M-M-M  8f M-D-D-L  9f L-D-D-M 10f M-M-M-L 11f L-M-M-M 12f M-M-M-M 13f L-D-D-L 14f M-M-M-M 15f L-L-L-L 16f M-M-M-M-M 17f M-D-M-D-L 18f M-D-L-D-M 19f L-D-M-D-M 20f M-M-M-M-L 21f M-M-L-M-M 22f L-M-M-M-M  1g A-K-K  2g A-A-K  3g A-D-K  4g K-D-A  5g K-A-A  6g K-K-K  7g A-A-A  8g A-D-D-K  9g K-D-D-A 10g A-A-A-K 11g K-A-A-A 12g A-A-A-A 13g K-D-D-K 14g A-A-A-A 15g K-K-K-K 16g A-A-A-A-A 17g A-D-A-D-K 18g A-D-K-D-A 19g K-D-A-D-A 20g A-A-A-A-K 21g A-A-K-A-A 22g K-A-A-A-A  1h E-K-K  2h E-E-K  3h E-D-K  4h K-D-E  5h K-E-E  6h K-K-K  7h E-E-E  8h E-D-D-K  9h K-D-D-E 10h E-E-E-K 11h K-E-E-E 12h E-E-E-E 13h K-D-D-K 14h E-E-E-E 15h K-K-K-K 16h E-E-E-E-E 17h E-D-E-D-K 18h E-D-K-D-E 19h K-D-E-D-E 20h E-E-E-E-K 21h E-E-K-E-E 22h K-E-E-E-E  1i M-K-K  2i M-M-K  3i M-D-K  4i K-D-M  5i K-M-M  6i K-K-K  7i M-M-M  8i M-D-D-K  9i K-D-D-M 10i M-M-M-K 11i K-M-M-M 12i M-M-M-M 13i K-D-D-K 14i M-M-M-M 15i K-K-K-K 16i M-M-M-M-M 17i M-D-M-D-K 18i M-D-K-D-M 19i K-D-M-D-M 20i M-M-M-M-K 21i M-M-K-M-M 22i K-M-M-M-M  1j A-L-K  2j M-E-K  3j L-D-K  4j K-D-A  5j B-M-E  6j K-L-L  7j E-M-E  8j E-D-D-M  9j M-D-D-E 10j E-M-E-B 11j B-E-E-M 12j E-E-E-M 13j K-L-D-K 14j E-M-E-M 15j K-L-L-K 16j E-E-M-E-E 17j E-D-M-D-K 18j E-D-K-D-M 19j B-D-A-D-A 20j E-M-E-E-L 21j E-E-K-M-M 22j B-E-M-E-A  1k K-D-K-D-K  1k A-K-L  2k M-E-L  3k K-D-L  4k L-D-K  5k L-M-E  6k L-K-L  7k M-E-M  8k K-D-D-L  9k L-D-K-E 10k E-M-E-L 11k L-E-E-M 12k M-E-E-E 13k L-K-D-L 14k M-EM-E 15k L-K-L-K 16k M-E-E-E-M 17k E-D-M-D-L 18k E-D-L-D-M 19k L-D-A-D-A 20k E-M-M-E-L 21k E-E-L-M-M 22k L-E-A-M-A  1l E-L-K  2l E-M-K  3l B-D-K  4l K-B-L  5l K-M-E  6l L-K-K  7l M-E-E  8l L-D-D-K  9l K-D-L-E 10l E-M-E-K 11l K-E-E-M 12l E-M-E-E 13l K-D-L-K 14l E-E-M-E 15l K-L-K-K 16l E-E-M-M-E 17l M-D-E-D-K 18l M-D-K-D-E 19l K-D-A-D-A 20l M-E-E-E-K 21l E-M-K-E-E 22l K-E-A-A-A

In the above table, “A” represents a nucleoside comprising a 2′-substituted sugar moiety; “B” represents a bicyclic nucleoside; “D” represents a 2′-deoxynucleoside; “K” represents a constrained ethyl nucleoside; “L” represents an LNA nucleoside; “E” represents a 2′-MOE nucleoside; and “M” represents a 2′-OMe nucleoside.

In certain embodiments, an oligonucleotide comprises any 5′-wing motif provided herein. In certain such embodiments, the oligonucleotide is a 5′-hemimer (does not comprise a 3′-wing). In certain embodiments, such an oligonucleotide is a gapmer. In certain such embodiments, the 3′-wing of the gapmer may comprise any sugar modification motif.

In certain embodiments, the 5′-wing of a gapmer has a sugar motif selected from among those listed in the following non-limiting tables:

TABLE 2 Certain 5′-Wing Sugar Motifs AAAAA AAAAB AAAAC AAABA AAABB AAABC AAACA AAACB AAACC AABAA AABAB AABAC AABBA AABBB AABBC AABCA AABCB AABCC   AACAA AACAB AACAC AACBA AACBB AACBC AACCA AACCB AACCC ABAAA ABAAB ABAAC ABABA ABABB ABABC ABACA ABACB ABACC ABBAA ABBAB ABBAC ABBBA ABBBB ABBBC ABBCA ABBCB ABBCC ABCAA ABCAB ABCAC ABCBA ABCBB ABCBC ABCCA ABCCB ABCCC ACAAA ACAAB ACAAC ACABA ACABB ACABC ACACA ACACB ACACC ACBAA ACBAB ACBAC ACBBA ACBBB ACBBC ACBCA ACBCB ACBCC ACCAA ACCAB ACCAC ACCBA ACCBB ACCBC ACCCA ACCCB ACCCC BAAAA BAAAB BAAAC BAABA BAABB BAABC BAACA BAACB BAACC BABAA BABAB BABAC BABBA BABBB BABBC BABCA BABCB BABCC BACAA BACAB BACAC BACBA BACBB BACBC BACCA BACCB BACCC BBAAA BBAAB BBAAC BBABA BBABB BBABC BBACA BBACB BBACC BBBAA BBBAB BBBAC BBBBA BBBBB BBBBC BBBCA BBBCB BBBCC BBCAA BBCAB BBCAC BBCBA BBCBB BBCBC BBCCA BBCCB BBCCC BCAAA BCAAB BCAAC BCABA BCABB BCABC BCACA BCACB BCACC BCBAA BCBAB BCBAC BCBBA BCBBB BCBBC BCBCA BCBCB BCBCC BCCAA BCCAB BCCAC BCCBA BCCBB BCCBC BCCCA BCCCB BCCCC CAAAA CAAAB CAAAC CAABA CAABB CAABC CAACA CAACB CAACC CABAA CABAB CABAC CABBA CABBB CABBC CABCA CABCB CABCC CACAA CACAB CACAC CACBA CACBB CACBC CACCA CACCB CACCC CBAAA CBAAB CBAAC CBABA CBABB CBABC CBACA CBACC CBBAA CBBAB CBBAC CBBBA CBBBB CBBBC CBBCA CBBCB CBBCC CBCAA CBCAB CBCAC CBCBA CBCBB CBCBC CBCCA CBCCB CBCCC CCAAA CCAAB CCAAC CCABA CCABB CCABC CCACA CCACB CCACC CCBAA CCBAB CCBAC CCBBA CCBBB CCBBC CCBCA CCBCB CCBCC CCCAA CCCAB CCCAC CCCBA CCCBB CCCBC CCCCA CCCCB CCCCC

TABLE 3 Certain 5′-Wing Sugar Motifs   AAAAA AAAAB AAABA AAABB AABAA AABAB AABBA AABBB ABAAA ABAAB ABABA ABABB ABBAA ABBAB ABBBA ABBBB BAAAA BAAAB BAABA BAABB BABAA BABAB BABBA BABBB BBAAA BBAAB BBABA BBABB BBBAA BBBAB BBBBA BBBBB AAAA AAAB AAAC AABA AABB AABC AACA AACB BABC BACA BACB BACC BBAA BBAB BBAC BBBA BBBB BBBC BBCA BBCB BBCC BCAA BCAB BCAC ABCB ABCC ACAA ACAB ACAC ACBA ACBB ACBC ACCA ACCB ACCC BAAA BAAB BAAC BABA BABB AACC ABAA ABAB ABAC ABBA ABBB ABBC ABCA CBAB CBAC CBBA CBBB CBBC CBCA CBCB CBCC CCAA CCAB CCAC CCBA CCBB CCBC CCCA CCCB BCBA BCBB BCBC BCCA BCCB BCCC CAAA CAAB CAAC CABA CABB CABC CACA CACB CACC CBAA CCCC AAAA AAAB AABA AABB ABAA ABAB ABBA ABBB BAAA BAAB BABA BABB BBAA BBAB BBBA BBBB AAA AAB AAC ABA ABB ABC ACA ACB ACC BAA BAB BAC BBA BBB BBC BCA BCB BCC CAA CAB CAC CBA CBB CBC CCA CCB CCC AAA AAB ABA ABB BAA BAB BBA BBB AA AB AC BA BB BC CA CB CC AA AB BA

In certain embodiments, each A, each B, and each C located at the 3′-most 5′-wing nucleoside is a modified nucleoside. For example, in certain embodiments the 5′-wing motif is selected from among ABB, BBB, and CBB, wherein the underlined nucleoside represents the 3′-most 5′-wing nucleoside and wherein the underlined nucleoside is a modified nucleoside.

In certain embodiments, each A comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, each A comprises a modified sugar moiety. In certain embodiments, each A comprises a 2′-substituted sugar moiety. In certain embodiments, each A comprises a 2′-substituted sugar moiety selected from among F, ara-F, OCH₃ and O(CH₂)₂—OCH₃. In certain embodiments, each A comprises a bicyclic sugar moiety. In certain embodiments, each A comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each A comprises a modified nucleobase. In certain embodiments, each A comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne uridine nucleoside. In certain embodiments, each A comprises an HNA. In certain embodiments, each A comprises an F-HNA.

In certain embodiments, each B comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, each B comprises a modified sugar moiety. In certain embodiments, each B comprises a 2′-substituted sugar moiety. In certain embodiments, each B comprises a 2′-substituted sugar moiety selected from among F, (ara)-F, OCH₃ and O(CH₂)₂—OCH₃. In certain embodiments, each B comprises a bicyclic sugar moiety. In certain embodiments, each B comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each B comprises a modified nucleobase. In certain embodiments, each B comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne urindine nucleoside. In certain embodiments, each B comprises an HNA. In certain embodiments, each B comprises an F-HNA.

In certain embodiments, each C comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, each C comprises a modified sugar moiety. In certain embodiments, each C comprises a 2′-substituted sugar moiety. In certain embodiments, each C comprises a 2′-substituted sugar moiety selected from among F, (ara)-F, OCH₃ and O(CH₂)₂—OCH₃. In certain embodiments, each C comprises a 5′-substituted sugar moiety. In certain embodiments, each C comprises a 5′-substituted sugar moiety selected from among 5′-Me, and 5′-(R)-Me. In certain embodiments, each C comprises a bicyclic sugar moiety. In certain embodiments, each C comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each C comprises a modified nucleobase. In certain embodiments, each C comprises a modified nucleobase selected from among 2-thio-thymidine and 5-propyne uridine. In certain embodiments, each C comprises a 2-thio-thymidine nucleoside. In certain embodiments, each C comprises an HNA. In certain embodiments, each C comprises an F-HNA.

In certain embodiments, at least one of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety, and the other comprises a 2′-substituted sugar moiety. In certain embodiments, at least one of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety, and the other comprises a bicyclic sugar moiety.

In certain embodiments, at least one of A or B comprises a bicyclic sugar moiety, and the other comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside and the other of A or B comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-MOE sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-MOE sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside and the other of A or B comprises a 2′-MOE sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-F sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-F sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside and the other of A or B comprises a 2′-F sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside and the other of A or B comprises a 2′-(ara)-F sugar moiety.

In certain embodiments, at least one of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety, and the other comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is an unmodified 2′-deoxyfuranose sugar moiety and the other of A or B comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is an unmodified 2′-deoxyfuranose sugar moiety and the other of A or B comprises a 2′-MOE sugar moiety. In certain embodiments, one of A or B is an unmodified 2′-deoxyfuranose sugar moiety and the other of A or B comprises a 2′-F sugar moiety. In certain embodiments, one of A or B is an unmodified 2′-deoxyfuranose sugar moiety and the other of A or B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, at least one of A or B comprises a bicyclic sugar moiety, and the other comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside and the other of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside and the other of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety.

In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2′-substituted sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2′-substituted sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2′-substituted sugar moiety. In certain embodiments, A is an α-L-LNA nucleoside and B comprises a 2′-substituted sugar moiety.

In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2′-MOE sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2′-MOE sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2′-MOE sugar moiety. In certain embodiments, A is an α-L-LNA nucleoside and B comprises a 2′-MOE sugar moiety.

In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2′-F sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2′-F sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2′-F sugar moiety. In certain embodiments, A is an α-L-LNA nucleoside and B comprises a 2′-F sugar moiety.

In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, A is an α-L-LNA nucleoside and B comprises a 2′-(ara)-F sugar moiety.

In certain embodiments, B comprises a bicyclic sugar moiety, and A comprises a 2′-MOE sugar moiety. In certain embodiments, B is an LNA nucleoside and A comprises a 2′-MOE sugar moiety. In certain embodiments, B is a cEt nucleoside and A comprises a 2′-MOE sugar moiety. In certain embodiments, B is an α-L-LNA nucleoside and A comprises a 2′-MOE sugar moiety.

In certain embodiments, B comprises a bicyclic sugar moiety, A comprises a 2′-MOE sugar moiety, and C comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, B is an LNA nucleoside, A comprises a 2′-MOE sugar moiety, and C comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, B is a cEt nucleoside, A comprises a 2′-MOE sugar moiety, and C comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, B is an α-L-LNA nucleoside and A comprises a 2′-MOE sugar moiety.

In certain embodiments, B comprises a bicyclic sugar moiety, and A comprises a 2′-F sugar moiety. In certain embodiments, B is an LNA nucleoside and A comprises a 2′-F sugar moiety. In certain embodiments, B is a cEt nucleoside and A comprises a 2′-F sugar moiety. In certain embodiments, B is an α-L-LNA nucleoside and A comprises a 2′-F sugar moiety.

In certain embodiments, B comprises a bicyclic sugar moiety, and A comprises a 2′-(ara)-F sugar moiety. In certain embodiments, B is an LNA nucleoside and A comprises a 2′-(ara)-F sugar moiety. In certain embodiments, B is a cEt nucleoside and A comprises a 2′-(ara)-F sugar moiety. In certain embodiments, B is an α-L-LNA nucleoside and A comprises a 2′-(ara)-F sugar moiety.

In certain embodiments, at least one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-substituted sugar moiety and C comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and C comprises a modified nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a modified nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a modified nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a modified nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-substituted sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and C comprises a 2-thio-thymidine nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 2-thio-thymidine nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 2-thio-thymidine nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises 2-thio-thymidine nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5-propyne uridine nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5-propyne uridine nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a HNA sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a sugar HNA surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a HNA sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a F-HNA sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a F-HNA sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a F-HNA sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-Me DNA sugar moiety.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety.

In certain embodiments, at least two of A, B or C comprises a 2′-substituted sugar moiety, and the other comprises a bicyclic sugar moiety. In certain embodiments, at least two of A, B or C comprises a bicyclic sugar moiety, and the other comprises a 2′-substituted sugar moiety. In certain embodiments, at least two of A, B or C comprises a 2′-substituted sugar moiety, and the other comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, at least two of A, B or C comprises a bicyclic sugar moiety, and the other comprises an unmodified 2′-deoxyfuranose sugar moiety.

Certain 3′-Wings

In certain embodiments, the 3′-wing of a gapmer consists of 1 to 5 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 2 to 5 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 3 to 5 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 4 or 5 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 1 to 4 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 1 to 3 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 1 or 2 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 2 to 4 linked nucleoside. In certain embodiments, the 3′-wing of a gapmer consists of 2 or 3 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 3 or 4 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 1 nucleoside. In certain embodiments, the 3′-wing of a gapmer consists of 2 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 3linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 4 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 5 linked nucleosides.

In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a bicyclic nucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a constrained ethyl nucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a LNA nucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least one non-bicyclic modified nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least two non-bicyclic modified nucleosides. In certain embodiments, the 3′-wing of a gapmer comprises at least three non-bicyclic modified nucleosides. In certain embodiments, the 3′-wing of a gapmer comprises at least four non-bicyclic modified nucleosides. In certain embodiments, the 3′-wing of a gapmer comprises at least one 2′-substituted nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one 2′-MOE nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one 2′-OMe nucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a non-bicyclic modified nucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a 2′-substituted nucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a 2′-MOE nucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a 2′-OMe nucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least one 2′-deoxynucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a 2′-deoxynucleoside. In a certain embodiments, the 3′-wing of a gapmer comprises at least one ribonucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a ribonucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-substituted nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-MOE nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-OMe nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-substituted nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-MOE nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-OMe nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside and at least one 2′-substituted nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside and at least one 2′-MOE nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside and at least one 2′-OMe nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside and at least one 2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside, at least one non-bicyclic modified nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one non-bicyclic modified nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside, at least one non-bicyclic modified nucleoside, and at least one 2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside, at least one 2′-substituted nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one 2′-substituted nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside, at least one 2′-substituted nucleoside, and at least one 2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside, at least one 2′-MOE nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one 2′-MOE nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside, at least one 2′-MOE nucleoside, and at least one 2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside, at least one 2′-OMe nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one 2′-OMe nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside, at least one 2′-OMe nucleoside, and at least one 2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer has a sugar motif selected from among those listed in the following non-limiting table:

TABLE 4 Certain 3′-Wing Sugar Motifs 3′-wing sugar motif # motif  1a B-B-A  2a B-B-B  3a A-A-B  4a B-A-B  5a B-A-B-A  6a B-B-B-A  7a B-D-B-A  8a B-B-B-B  9a B-D-D-B 10a A-B-B-A  1b B-B-E  2b B-B-B  3b E-E-B  4b B-E-B  5b B-E-B-E  6b B-B-B-E  7b B-D-B-E  8b B-B-B-B  9b B-D-D-B 10b E-B-B-E  1c B-B-M  2c B-B-B  3c M-M-B  4c B-M-B  5c B-M-B-M  6c B-B-B-M  7c B-D-B-M  8c B-B-B-B  9c B-D-D-B 10c M-B-B-M  1d L-L-A  2d L-L-L  3d A-A-L  4d L-A-L  5d L-A-L-A  6d L-L-L-A  7d L-D-L-A  8d L-L-L-L  9d L-D-D-L 10d A-L-L-A  1e L-L-E  2e L-L-L  3e E-E-L  4e L-E-L  5e L-E-L-E  6e L-L-L-E  7e L-D-L-E  8e L-L-L-L  9e L-D-D-L 10e E-L-L-E  1f L-L-M  2f L-L-L  3f M-M-L  4f L-M-L  5f L-M-L-M  6f L-L-L-M  7f L-D-L-M  8f L-L-L-L  9f L-D-D-L 10f M-L-L-M  1g K-K-A  2g K-K-K  3g A-A-K  4g K-A-K  5g K-A-K-A  6g K-K-K-A  7g K-D-K-A  8g K-K-K-K  9g K-D-D-K 10g A-K-K-A  1h K-K-E  2h K-K-K  3h E-E-K  4h K-E-K  5h K-E-K-E  6h K-K-K-E  7h K-D-K-E  8h K-K-K-K  9h K-D-D-K 10h E-K-K-E  1i K-K-M  2i K-K-K  3i M-M-K  4i K-M-K  5i K-M-K-M  6i K-K-K-M  7i K-D-K-M  8i K-K-K-K  9i K-D-D-K 10i M-K-K-M  1j K-K-A  2j K-L-L  3j E-M-B  4j K-A-L  5j K-A-L-A  6j K-L-K-A  7j L-D-K-A  8j B-K-L-B  9j K-D-D-B 10j A-K-B-A  1k L-K-A  2k K-K-L  3k E-M-L  4k L-A-K  5k L-A-K-A  6k K-K-L-A  7k K-D-L-A  8k K-L-L-L  9k K-D-D-L 10k A-K-L-A  1l K-L-E  2l K-L-K  3l E-K-K  4l L-E-K  5l K-E-L-E  6l K-L-K-A  7l K-D-L-E  8l K-K-L-K  9l L-D-D-K 10l A-B-K-A 1m E-E

In the above table, “A” represents a nucleoside comprising a 2′-substituted sugar moiety; “B” represents a bicyclic nucleoside; “D” represents a 2′-deoxynucleoside; “K” represents a constrained ethyl nucleoside; “L” represents an LNA nucleoside; “E” represents a 2′-MOE nucleoside; and “M” represents a 2′-OMe nucleoside.

In certain embodiments, an oligonucleotide comprises any 3′-wing motif provided herein. In certain such embodiments, the oligonucleotide is a 3′-hemimer (does not comprise a 5′-wing). In certain embodiments, such an oligonucleotide is a gapmer. In certain such embodiments, the 5′-wing of the gapmer may comprise any sugar modification motif.

In certain embodiments, the 5′-wing of a gapmer has a sugar motif selected from among those listed in the following non-limiting tables:

TABLE 5 Certain 3′-Wing Sugar Motifs AAAAA AAAAB AAAAC AAABA AAABB AAABC AAACA AAACB AAACC AABAA AABAB AABAC AABBA AABBB AABBC AABCA AABCB AABCC AACAA AACAB AACAC AACBA AACBB AACBC AACCA AACCB AACCC ABAAA ABAAB ABAAC ABABA ABABB ABABC ABACA ABACB ABACC ABBAA ABBAB ABBAC ABBBA ABBBB ABBBC ABBCA ABBCB ABBCC ABCAA ABCAB ABCAC ABCBA ABCBB ABCBC ABCCA ABCCB ABCCC ACAAA ACAAB ACAAC ACABA ACABB ACABC ACACA ACACB ACACC ACBAA ACBAB ACBAC ACBBA ACBBB ACBBC ACBCA ACBCB ACBCC ACCAA ACCAB ACCAC ACCBA ACCBB ACCBC ACCCA ACCCB ACCCC BAAAA BAAAB BAAAC BAABA BAABB BAABC BAACA BAACB BAACC BABAA BABAB BABAC BABBA BABBB BABBC BABCA BABCB BABCC BACAA BACAB BACAC BACBA BACBB BACBC BACCA BACCB BACCC BBAAA BBAAB BBAAC BBABA BBABB BBABC BBACA BBACB BBACC BBBAA BBBAB BBBAC BBBBA BBBBB BBBBC BBBCA BBBCB BBBCC BBCAA BBCAB BBCAC BBCBA BBCBB BBCBC BBCCA BBCCB BBCCC BCAAA BCAAB BCAAC BCABA BCABB BCABC BCACA BCACB BCACC BCBAA BCBAB BCBAC BCBBA BCBBB BCBBC BCBCA BCBCB BCBCC BCCAA BCCAB BCCAC BCCBA BCCBB BCCBC BCCCA BCCCB BCCCC CAAAA CAAAB CAAAC CAABA CAABB CAABC CAACA CAACB CAACC CABAA CABAB CABAC CABBA CABBB CABBC CABCA CABCB CABCC CACAA CACAB CACAC CACBA   CACBB CACBC CACCA CACCB CACCC CBAAA CBAAB CBAAC CBABA CBABB CBABC CBACA CBACC CBBAA CBBAB CBBAC CBBBA CBBBB CBBBC CBBCA CBBCB CBBCC CBCAA CBCAB CBCAC CBCBA CBCBB CBCBC CBCCA CBCCB CBCCC CCAAA CCAAB CCAAC CCABA CCABB CCABC CCACA CCACB CCACC CCBAA CCBAB CCBAC CCBBA CCBBB CCBBC CCBCA CCBCB CCBCC CCCAA CCCAB CCCAC CCCBA CCCBB CCCBC CCCCA CCCCB CCCCC

TABLE 6 Certain 3′-Wing Sugar Motifs AAAAA AAAAB AAABA AAABB AABAA AABAB AABBA AABBB ABAAA ABAAB ABABA ABABB ABBAA ABBAB ABBBA ABBBB BAAAA BAAAB BAABA BAABB BABAA BABAB BABBA BABBB BBAAA BBAAB BBABA BBABB BBBAA BBBAB BBBBA BBBBB AAAA AAAB AAAC AABA AABB AABC AACA AACB BABC BACA BACB BACC BBAA BBAB BBAC BBBA BBBB BBBC BBCA BBCB BBCC BCAA BCAB BCAC ABCB ABCC ACAA ACAB ACAC ACBA ACBB ACBC ACCA ACCB ACCC BAAA BAAB BAAC BABA BABB AACC ABAA ABAB ABAC ABBA ABBB ABBC ABCA CBAB CBAC CBBA CBBB CBBC CBCA CBCB CBCC CCAA CCAB CCAC CCBA CCBB CCBC CCCA CCCB BCBA BCBB BCBC BCCA BCCB BCCC CAAA CAAB CAAC CABA CABB CABC CACA CACB CACC CBAA CCCC AAAA AAAB AABA AABB ABAA ABAB ABBA ABBB BAAA BAAB BABA BABB BBAA BBAB BBBA BBBB AAA AAB AAC ABA ABB ABC ACA ACB ACC BAA BAB BAC BBA BBB BBC BCA BCB BCC CAA CAB CAC CBA CBB CBC CCA CCB CCC AAA AAB ABA ABB BAA BAB BBA BBB AA AB AC BA BB BC CA   CB CC AA AB BA

In certain embodiments, each A, each B, and each C located at the 5′-most 3′-wing region nucleoside is a modified nucleoside. For example, in certain embodiments the 3′-wing motif is selected from among ABB, BBB, and CBB, wherein the underlined nucleoside represents the the 5′-most 3′-wing region nucleoside and wherein the underlined nucleoside is a modified nucleoside.

In certain embodiments, each A comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, each A comprises a modified sugar moiety. In certain embodiments, each A comprises a 2′-substituted sugar moiety. In certain embodiments, each A comprises a 2′-substituted sugar moiety selected from among F, ara-F, OCH₃ and O(CH₂)₂—OCH₃. In certain embodiments, each A comprises a bicyclic sugar moiety. In certain embodiments, each A comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each A comprises a modified nucleobase. In certain embodiments, each A comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne uridine nucleoside.

In certain embodiments, each B comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, each B comprises a modified sugar moiety. In certain embodiments, each B comprises a 2′-substituted sugar moiety. In certain embodiments, each B comprises a 2′-substituted sugar moiety selected from among F, (ara)-F, OCH₃ and O(CH₂)₂—OCH₃. In certain embodiments, each B comprises a bicyclic sugar moiety. In certain embodiments, each B comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each B comprises a modified nucleobase. In certain embodiments, each B comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne urindine nucleoside.

In certain embodiments, each C comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, each C comprises a modified sugar moiety. In certain embodiments, each C comprises a 2′-substituted sugar moiety. In certain embodiments, each C comprises a 2′-substituted sugar moiety selected from among F, (ara)-F, OCH₃ and O(CH₂)₂—OCH₃. In certain embodiments, each C comprises a 5′-substituted sugar moiety. In certain embodiments, each C comprises a 5′-substituted sugar moiety selected from among 5′-Me, and 5′-(R)-Me. In certain embodiments, each C comprises a bicyclic sugar moiety. In certain embodiments, each C comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each C comprises a modified nucleobase. In certain embodiments, each C comprises a modified nucleobase selected from among 2-thio-thymidine and 5-propyne uridine. In certain embodiments, each C comprises a 2-thio-thymidine nucleoside.

In certain embodiments, at least one of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety, and the other comprises a 2′-substituted sugar moiety. In certain embodiments, at least one of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety, and the other comprises a bicyclic sugar moiety.

In certain embodiments, at least one of A or B comprises a bicyclic sugar moiety, and the other comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside and the other of A or B comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-MOE sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-MOE sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside and the other of A or B comprises a 2′-MOE sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-F sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-F sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside and the other of A or B comprises a 2′-F sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside and the other of A or B comprises a 2′-(ara)-F sugar moiety.

In certain embodiments, at least one of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety, and the other comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is an unmodified 2′-deoxyfuranose sugar moiety and the other of A or B comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is an unmodified 2′-deoxyfuranose sugar moiety and the other of A or B comprises a 2′-MOE sugar moiety. In certain embodiments, one of A or B is an unmodified 2′-deoxyfuranose sugar moiety and the other of A or B comprises a 2′-F sugar moiety. In certain embodiments, one of A or B is an unmodified 2′-deoxyfuranose sugar moiety and the other of A or B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, at least one of A or B comprises a bicyclic sugar moiety, and the other comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside and the other of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside and the other of A or B comprises an unmodified 2′-deoxyfuranose sugar moiety.

In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2′-substituted sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2′-substituted sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2′-substituted sugar moiety. In certain embodiments, A is an α-L-LNA nucleoside and B comprises a 2′-substituted sugar moiety.

In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2′-MOE sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2′-MOE sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2′-MOE sugar moiety. In certain embodiments, A is an α-L-LNA nucleoside and B comprises a 2′-MOE sugar moiety.

In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2′-F sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2′-F sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2′-F sugar moiety. In certain embodiments, A is an α-L-LNA nucleoside and B comprises a 2′-F sugar moiety.

In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, A is an α-L-LNA nucleoside and B comprises a 2′-(ara)-F sugar moiety.

In certain embodiments, B comprises a bicyclic sugar moiety, and A comprises a 2′-MOE sugar moiety. In certain embodiments, B is an LNA nucleoside and A comprises a 2′-MOE sugar moiety. In certain embodiments, B is a cEt nucleoside and A comprises a 2′-MOE sugar moiety. In certain embodiments, B is an α-L-LNA nucleoside and A comprises a 2′-MOE sugar moiety.

In certain embodiments, B comprises a bicyclic sugar moiety, A comprises a 2′-MOE sugar moiety, and C comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, B is an LNA nucleoside, A comprises a 2′-MOE sugar moiety, and C comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, B is a cEt nucleoside, A comprises a 2′-MOE sugar moiety, and C comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, B is an α-L-LNA nucleoside and A comprises a 2′-MOE sugar moiety.

In certain embodiments, B comprises a bicyclic sugar moiety, and A comprises a 2′-F sugar moiety. In certain embodiments, B is an LNA nucleoside and A comprises a 2′-F sugar moiety. In certain embodiments, B is a cEt nucleoside and A comprises a 2′-F sugar moiety. In certain embodiments, B is an α-L-LNA nucleoside and A comprises a 2′-F sugar moiety.

In certain embodiments, B comprises a bicyclic sugar moiety, and A comprises a 2′-(ara)-F sugar moiety. In certain embodiments, B is an LNA nucleoside and A comprises a 2′-(ara)-F sugar moiety. In certain embodiments, B is a cEt nucleoside and A comprises a 2′-(ara)-F sugar moiety. In certain embodiments, B is an α-L-LNA nucleoside and A comprises a 2′-(ara)-F sugar moiety.

In certain embodiments, at least one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-substituted sugar moiety and C comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and C comprises a modified nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a modified nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a modified nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a modified nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-substituted sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and C comprises a 2-thio-thymidine nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 2-thio-thymidine nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 2-thio-thymidine nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises 2-thio-thymidine nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5-propyne uridine nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5-propyne uridine nucleobase.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a HNA sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a sugar HNA surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a HNA sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B. comprises a 2′-MOE sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a F-HNA sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a F-HNA sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a F-HNA sugar surrogate.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-Me DNA sugar moiety.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-Me DNA sugar moiety.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety.

In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and C comprises a 5′-(R)-Me DNA sugar moiety.

In certain embodiments, at least two of A, B or C comprises a 2′-substituted sugar moiety, and the other comprises a bicyclic sugar moiety. In certain embodiments, at least two of A, B or C comprises a bicyclic sugar moiety, and the other comprises a 2′-substituted sugar moiety.

In certain embodiments, at least two of A, B or C comprises a 2′-substituted sugar moiety, and the other comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, at least two of A, B or C comprises a bicyclic sugar moiety, and the other comprises an unmodified 2′-deoxyfuranose sugar moiety.

Certain Gaps

In certain embodiments, the gap of a gapmer consists of 6 to 20 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 15 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 12 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 10 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 9 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 8 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 or 7 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 7 to 10 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 7 to 9 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 7 or 8 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 8 to 10 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 8 or 9 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 7 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 8 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 9 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 10 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 11 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 12 linked nucleosides.

In certain embodiments, each nucleotide of the gap of a gapmer is a 2′-deoxynucleoside. In certain embodiments, the gap comprises one or more modified nucleosides. In certain embodiments, each nucleotide of the gap of a gapmer is a 2′-deoxynucleoside or is a modified nucleoside that is “DNA-like.” In such embodiments, “DNA-like” means that the nucleoside has similar characteristics to DNA, such that a duplex comprising the gapmer and an RNA molecule is capable of activating RNase H. For example, under certain conditions, 2′- fluoro (arabino) nucleosides (also referred to as FANA) have been shown to support RNase H activation, and thus is DNA-like. In certain embodiments, one or more nucleosides of the gap of a gapmer is not a 2′-deoxynucleoside and is not DNA-like. In certain such embodiments, the gapmer nonetheless supports RNase H activation (e.g., by virtue of the number or placement of the non-DNA nucleosides).

Certain Gapmer Motifs

In certain embodiments, a gapmer comprises a 5′-wing, a gap, and a 3′ wing, wherein the 5′-wing, gap, and 3′ wing are independently selected from among those discussed above. For example, in certain embodiments, a gapmer has a 5′-wing selected from any of the 5′-wing motifs in Tables 1, 2, and 3 above and a 3′-wing selected from any of the 3′-wing motifs in Tables, 4, 5, and 6. For example, in certain embodiments, a gapmer has a 5′-wing, a gap, and a 3′-wing having features selected from among those listed in the following non-limiting table:

TABLE 7 Certain Gapmer Sugar Motifs Gapmer motif # 5-wing Gap 3′-wing 1 At least one non-bicyclic All 2′-deoxynucleosides At least one bicyclic modified nucleoside nucleoside 2 At least one non-bicyclic All 2′-deoxynucleosides At least one LNA nucleoside modified nucleoside 3 At least one non-bicyclic All 2′-deoxynucleosides At least one cEt nucleoside modified nucleoside 4 At least one 2′-substituted All 2′-deoxynucleosides At least one bicyclic nucleoside nucleoside 5 At least one 2′-substituted All 2′-deoxynucleosides At least one LNA nucleoside nucleoside 6 At least one 2′-substituted All 2′-deoxynucleosides At least one cEt nucleoside nucleoside 7 At least one 2′-MOE nucleoside All 2′-deoxynucleosides At least one bicyclic nucleoside 8 At least one 2′-MOE nucleoside All 2′-deoxynucleosides At least one LNA nucleoside 9 At least one 2′-MOE nucleoside All 2′-deoxynucleosides At least one cEt nucleoside 10 At least one 2′-OMe nucleoside All 2′-deoxynucleosides At least one bicyclic nucleoside 11 At least one 2′-OMe nucleoside All 2′-deoxynucleosides At least one LNA nucleoside 12 At least one 2′-OMe nucleoside All 2′-deoxynucleosides At least one cEt nucleoside 13 At least one 2′-deoxynucleoside All 2′-deoxynucleosides At least one bicyclic nucleoside 14 At least one 2′-deoxynucleoside All 2′-deoxynucleosides At least one LNA nucleoside 15 At least one 2′-deoxynucleoside All 2′-deoxynucleosides At least one cEt nucleoside 16 At least one bicyclic nucleoside All 2′-deoxynucleosides At least one non-bicyclic modified nucleoside 17 At least one LNA nucleoside All 2′-deoxynucleosides At least one non-bicyclic modified nucleoside 18 At least one cEt nucleoside All 2′-deoxynucleosides At least one non-bicyclic modified nucleoside 19 At least one bicyclic nucleoside All 2′-deoxynucleosides At least one 2′-substituted nucleoside 20 At least one LNA nucleoside All 2′-deoxynucleosides At least one 2′-substituted nucleoside 21 At least one cEt nucleoside All 2′-deoxynucleosides At least one 2′-substituted nucleoside 22 At least one bicyclic nucleoside All 2′-deoxynucleosides At least one 2′-MOE nucleoside 23 At least one LNA nucleoside All 2′-deoxynucleosides At least one 2′-MOE nucleoside 24 At least one cEt nucleoside All 2′-deoxynucleosides At least one 2′-MOE nucleoside 25 At least one bicyclic nucleoside All 2′-deoxynucleosides At least one 2′-OMe nucleoside 26 At least one LNA nucleoside All 2′-deoxynucleosides At least one 2′-OMe nucleoside 27 At least one cEt nucleoside All 2′-deoxynucleosides At least one 2′-OMe nucleoside 28 At least one bicyclic nucleoside All 2′-deoxynucleosides At least one 2′- deoxynucleoside 29 At least one LNA nucleoside All 2′-deoxynucleosides At least one 2′- deoxynucleoside 30 At least one cEt nucleoside All 2′-deoxynucleosides At least one 2′- deoxynucleoside 31 At least one bicyclic nucleoside All 2′-deoxynucleosides At least one bicyclic and at least one 2′-substituted nucleoside and at least one 2′- nucleoside substituted nucleoside 32 At least one bicyclic nucleoside All 2′-deoxynucleosides At least two bicyclic and at least one 2′-substituted nucleosides nucleoside 33 At least one cEt nucleoside and All 2′-deoxynucleosides At least one bicyclic at least one 2′-substituted nucleoside and at least one 2′- nucleoside substituted nucleoside 34 At least one cEt nucleoside and All 2′-deoxynucleosides At least two bicyclic at least one 2′-substituted nucleosides nucleoside 35 At least one LNA nucleoside All 2′-deoxynucleosides At least one bicyclic and at least one 2′-substituted nucleoside and at least one 2′- nucleoside substituted nucleoside 36 At least one LNA nucleoside All 2′-deoxynucleosides At least two bicyclic and at least one 2′-substituted nucleosides nucleoside 37 At least one bicyclic nucleoside All 2′-deoxynucleosides At least one LNA nucleoside and at least one 2′-substituted and at least one 2′-substituted nucleoside nucleoside 38 At least one bicyclic nucleoside All 2′-deoxynucleosides At least two LNA nucleosides and at least one 2′-substituted nucleoside 39 At least one cEt nucleoside and All 2′-deoxynucleosides At least one LNA nucleoside at least one 2′-substituted and at least one 2′-substituted nucleoside nucleoside 40 At least one cEt nucleoside and All 2′-deoxynucleosides At least two LNA nucleosides at least one 2′-substituted nucleoside 41 At least one LNA nucleoside and All 2′-deoxynucleosides At least one LNA nucleoside at least one 2′-substituted and at least one 2′-substituted nucleoside nucleoside 42 At least one LNA nucleoside and All 2′-deoxynucleosides At least two LNA nucleosides at least one 2′-substituted nucleoside 43 At least one bicyclic nucleoside All 2′-deoxynucleosides At least one bicyclic and at least one 2′- nucleoside and at least one 2′- deoxynucleoside substituted nucleoside 44 At least one bicyclic nucleoside All 2′-deoxynucleosides At least two bicyclic and at least one 2′- nucleosides deoxynucleoside 45 At least one cEt nucleoside and All 2′-deoxynucleosides At least one bicyclic at least one 2′-deoxynucleoside nucleoside and at least one 2′- substituted nucleoside 46 At least one cEt nucleoside and All 2′-deoxynucleosides At least two bicyclic at least one 2′-deoxynucleoside nucleosides 47 At least one LNA nucleoside and All 2′-deoxynucleosides At least one bicyclic at least one 2′-deoxynucleoside nucleoside and at least one 2′- substituted nucleoside 48 At least one LNA nucleoside and All 2′-deoxynucleosides At least two bicyclic at least one 2′-deoxynucleoside nucleosides 49 At least one bicyclic nucleoside All 2′-deoxynucleosides At least one LNA nucleoside and at least one 2′- and at least one 2′-substituted deoxynucleoside nucleoside 50 At least one bicyclic nucleoside All 2′-deoxynucleosides At least two LNA nucleosides and at least one 2′- deoxynucleoside 51 At least one cEt nucleoside and All 2′-deoxynucleosides At least one LNA nucleoside at least one 2′-deoxynucleoside and at least one 2′-substituted nucleoside 52 At least one cEt nucleoside and All 2′-deoxynucleosides At least two LNA nucleosides at least one 2′-deoxynucleoside 53 At least one LNA nucleoside and All 2′-deoxynucleosides At least one LNA nucleoside at least one 2′-deoxynucleoside and at least one 2′-substituted nucleoside 54 At least one LNA nucleoside and All 2′-deoxynucleosides At least two LNA nucleosides at least one 2′-deoxynucleoside 55 At least two 2′-substituted All 2′-deoxynucleosides At least one bicyclic nucleosides nucleoside and at least one 2′- substituted nucleoside 56 At least two 2′-substituted All 2′-deoxynucleosides At least two bicyclic nucleosides nucleosides 57 At least two 2′-substituted All 2′-deoxynucleosides At least one LNA nucleoside nucleosides and at least one 2′-substituted nucleoside 58 At least two 2′-substituted All 2′-deoxynucleosides At least two LNA nucleosides nucleosides

In certain embodiments, a gapmer comprises a 5′-wing, a gap, and a 3′ wing, wherein the 5′-wing, gap, and 3′ wing are independently selected from among those discussed above. For example, in certain embodiments, a gapmer has a 5′-wing, a gap, and a 3′-wing wherein the 5′-wing and the 3′-wing have features selected from among those listed in the tables above. In certain embodiments, any 5′-wing may be paired with any 3′-wing. In certain embodiments the 5′-wing may comprise ABBBB and the 3′-wing may comprise BBA. In certain embodiments the 5′-wing may comprise ACACA and the 3′-wing may comprise BB. For example, in certain embodiments, a gapmer has a 5′-wing, a gap, and a 3′-wing having features selected from among those listed in the following non-limiting table, wherein each motif is represented as (5′-wing)-(gap)-(3′-wing), wherein each number represents the number of linked nucleosides in each portion of the motif, for example, a 5-10-5 motif would have a 5′-wing comprising 5 nucleosides, a gap comprising 10 nucleosides, and a 3′-wing comprising 5 nucleosides:

TABLE 8 Certain Gapmer Sugar Motifs 2-10-2 3-10-2 4-10-2 5-10-2 2-10-3 3-10-3 4-10-3 5-10-3 2-10-4 3-10-4 4-10-4 5-10-4 2-10-5 3-10-5 4-10-5 5-10-5 2-9-2 3-9-2 4-9-2 5-9-2 2-9-3 3-9-3 4-9-3 5-9-3 2-9-4 3-9-4 4-9-4 5-9-4 2-9-5 3-9-5 4-9-5 5-9-5 2-11-2 3-11-2 4-11-2 5-11-2 2-11-3 3-11-3 4-11-3 5-11-3 2-11-4 3-11-4 4-11-4 5-11-4 2-11-5 3-11-5 4-11-5 5-11-5 2-8-2 3-8-2 4-8-2 5-8-2 2-8-3 3-8-3 4-8-3 5-8-3 2-8-4 3-8-4 4-8-4 5-8-4 2-8-5 3-8-5 4-8-5 5-8-5

In certain embodiments, gapmers have a motif described by Formula I as follows:

(A)_(m)-(B)_(n)-(J)_(p)-(B)_(r)-(J)_(t)-(D)_(g-h)-(J)_(v)-(B)_(w)-(J)_(x)-(B)_(y)-(A)_(z)

wherein:

each A is independently a 2′-substituted nucleoside;

each B is independently a bicyclic nucleoside;

each J is independently either a 2′-substituted nucleoside or a 2′-deoxynucleoside;

each D is a 2′-deoxynucleoside;

m is 0-4; n is 0-2; p is 0-2; r is 0-2; t is 0-2; v is 0-2; w is 0-4; x is 0-2; y is 0-2; z is 0-4; g is 6; and h is 14;

provided that:

at least one of m, n, and r is other than 0;

at least one of w and y is other than 0;

the sum of m, n, p, r, and t is from 2 to 5; and

the sum of v, w, x, y, and z is from 2 to 5.

In certain embodiments, one or more 2′-substituted nucleoside is a 2′-MOE nucleoside. In certain embodiments, one or more 2′-substituted nucleoside is a 2′-OMe nucleoside. In certain In certain embodiments, one or more bicyclic nucleoside is a cEt nucleoside. In certain embodiments, one or more bicyclic nucleoside is an LNA nucleoside.

In certain embodiments, a gapmer of Formula I has a motif selected from among gapmer motifs 1-58.

In certain embodiments, gapmers have a motif described by Formula II as follows:

(J)_(m)-(B)_(n)-(J)_(p)-(B)_(r)-(A)_(t)-(D)_(g)-(A)_(v)-(B)_(w)-(J)_(x)-(B)_(y)-(J)_(z)

wherein:

each A is independently a 2′-substituted nucleoside;

each B is independently a bicyclic nucleoside;

each J is independently either a 2′-substituted nucleoside or a 2′-deoxynucleoside;

each D is a 2′-deoxynucleoside;

m is 0-4; n is 0-2; p is 0-2; r is 0-2; t is 0-2; v is 0-2; w is 0-4; x is 0-2; y is 0-2; z is 0-4; g is 6-14;

provided that:

at least one of m, n, and r is other than 0;

at least one of w and y is other than 0;

the sum of m, n, p, r, and t is from 1 to 5; and

the sum of v, w, x, y, and z is from 1 to 5.

In certain embodiments, one or more 2′-substituted nucleoside is a 2′-MOE nucleoside. In certain embodiments, one or more 2′-substituted nucleoside is a 2′-OMe nucleoside. In certain embodiments, one or more bicyclic nucleoside is a cEt nucleoside. In certain embodiments, one or more bicyclic nucleoside is an LNA nucleoside.

In certain embodiments, each 2′-substituted nucleoside is a 2′-MOE nucleoside. In certain embodiments, each 2′-substituted nucleoside is a 2′-OMe nucleoside. In certain embodiments, each bicyclic nucleoside is a cEt nucleoside. In certain embodiments, each bicyclic nucleoside is an LNA nucleoside.

In certain embodiments, each A is the same 2′-substituted nucleoside. In certain embodiments, each B is the same bicyclic nucleoside. In certain embodiments each A is the same 2′-modified nucleoside and each B is the same bicyclic nucleoside. In certain embodiments, each J is a 2′-modified nucleoside. In certain embodiments each J is the same 2′-modified nucleoside. In certain embodiments, each J and each A is the same 2′-modified nucleoside.

In certain embodiments, a gapmer of Formula II has a motif selected from among gapmer motifs 1-58.

In certain embodiments, a gapmer comprises a 5′-wing, a gap, and a 3′ wing, independently selected from among those proved in the above tables, for example as provided in the following table:

TABLE 9 Certain Gapmer Sugar Motifs Gapmer 5-wing sugar motif 3′-wing sugar motif motif # (from table 1) Gap (from table 2) 59  1(a-i) All 2′-deoxynucleosides  1(a-i) 60  2(a-i) All 2′-deoxynucleosides  1(a-i) 61  3(a-i) All 2′-deoxynucleosides  1(a-i) 62  4(a-i) All 2′-deoxynucleosides  1(a-i) 63  5(a-i) All 2′-deoxynucleosides  1(a-i) 64  6(a-i) All 2′-deoxynucleosides  1(a-i) 65  7(a-i) All 2′-deoxynucleosides  1(a-i) 66  8(a-i) All 2′-deoxynucleosides  1(a-i) 67  9(a-i) All 2′-deoxynucleosides  1(a-i) 68 10(a-i) All 2′-deoxynucleosides  1(a-i) 69 11(a-i) All 2′-deoxynucleosides  1(a-i) 70 12(a-i) All 2′-deoxynucleosides  1(a-i) 71 13(a-i) All 2′-deoxynucleosides  1(a-i) 72 14(a-i) All 2′-deoxynucleosides  1(a-i) 73 15(a-i) All 2′-deoxynucleosides  1(a-i) 74 16(a-i) All 2′-deoxynucleosides  1(a-i) 75 17(a-i) All 2′-deoxynucleosides  1(a-i) 76 18(a-i) All 2′-deoxynucleosides  1(a-i) 77 19(a-i) All 2′-deoxynucleosides  1(a-i) 78 20(a-i) All 2′-deoxynucleosides  1(a-i) 79 21(a-i) All 2′-deoxynucleosides  1(a-i) 80 22(a-i) All 2′-deoxynucleosides  1(a-i) 81  1(a-i) All 2′-deoxynucleosides  2(a-i) 82  2(a-i) All 2′-deoxynucleosides  2(a-i) 83  3(a-i) All 2′-deoxynucleosides  2(a-i) 84  4(a-i) All 2′-deoxynucleosides  2(a-i) 85  5(a-i) All 2′-deoxynucleosides  2(a-i) 86  6(a-i) All 2′-deoxynucleosides  2(a-i) 87  7(a-i) All 2′-deoxynucleosides  2(a-i) 88  8(a-i) All 2′-deoxynucleosides  2(a-i) 89  9(a-i) All 2′-deoxynucleosides  2(a-i) 90 10(a-i) All 2′-deoxynucleosides  2(a-i) 91 11(a-i) All 2′-deoxynucleosides  2(a-i) 92 12(a-i) All 2′-deoxynucleosides  2(a-i) 93 13(a-i) All 2′-deoxynucleosides  2(a-i) 94 14(a-i) All 2′-deoxynucleosides  2(a-i) 94 15(a-i) All 2′-deoxynucleosides  2(a-i) 96 16(a-i) All 2′-deoxynucleosides  2(a-i) 97 17(a-i) All 2′-deoxynucleosides  2(a-i) 98 18(a-i) All 2′-deoxynucleosides  2(a-i) 99 19(a-i) All 2′-deoxynucleosides  2(a-i) 100 20(a-i) All 2′-deoxynucleosides  2(a-i) 101 21(a-i) All 2′-deoxynucleosides  2(a-i) 102 22(a-i) All 2′-deoxynucleosides  2(a-i) 103  1(a-i) All 2′-deoxynucleosides  3(a-i) 104  2(a-i) All 2′-deoxynucleosides  3(a-i) 105  3(a-i) All 2′-deoxynucleosides  3(a-i) 106  4(a-i) All 2′-deoxynucleosides  3(a-i) 107  5(a-i) All 2′-deoxynucleosides  3(a-i) 108  6(a-i) All 2′-deoxynucleosides  3(a-i) 109  7(a-i) All 2′-deoxynucleosides  3(a-i) 110  8(a-i) All 2′-deoxynucleosides  3(a-i) 111  9(a-i) All 2′-deoxynucleosides  3(a-i) 112 10(a-i) All 2′-deoxynucleosides  3(a-i) 113 11(a-i) All 2′-deoxynucleosides  3(a-i) 114 12(a-i) All 2′-deoxynucleosides  3(a-i) 115 13(a-i) All 2′-deoxynucleosides  3(a-i) 116 14(a-i) All 2′-deoxynucleosides  3(a-i) 117 15(a-i) All 2′-deoxynucleosides  3(a-i) 118 16(a-i) All 2′-deoxynucleosides  3(a-i) 119 17(a-i) All 2′-deoxynucleosides  3(a-i) 120 18(a-i) All 2′-deoxynucleosides  3(a-i) 121 19(a-i) All 2′-deoxynucleosides  3(a-i) 122 20(a-i) All 2′-deoxynucleosides  3(a-i) 123 21(a-i) All 2′-deoxynucleosides  3(a-i) 124 22(a-i) All 2′-deoxynucleosides  3(a-i) 125  1(a-i) All 2′-deoxynucleosides  4(a-i) 126  2(a-i) All 2′-deoxynucleosides  4(a-i) 127  3(a-i) All 2′-deoxynucleosides  4(a-i) 128  4(a-i) All 2′-deoxynucleosides  4(a-i) 129  5(a-i) All 2′-deoxynucleosides  4(a-i) 130  6(a-i) All 2′-deoxynucleosides  4(a-i) 131  7(a-i) All 2′-deoxynucleosides  4(a-i) 132  8(a-i) All 2′-deoxynucleosides  4(a-i) 133  9(a-i) All 2′-deoxynucleosides  4(a-i) 134 10(a-i) All 2′-deoxynucleosides  4(a-i) 135 11(a-i) All 2′-deoxynucleosides  4(a-i) 136 12(a-i) All 2′-deoxynucleosides  4(a-i) 137 13(a-i) All 2′-deoxynucleosides  4(a-i) 138 14(a-i) All 2′-deoxynucleosides  4(a-i) 139 15(a-i) All 2′-deoxynucleosides  4(a-i) 140 16(a-i) All 2′-deoxynucleosides  4(a-i) 141 17(a-i) All 2′-deoxynucleosides  4(a-i) 142 18(a-i) All 2′-deoxynucleosides  4(a-i) 143 19(a-i) All 2′-deoxynucleosides  4(a-i) 144 20(a-i) All 2′-deoxynucleosides  4(a-i) 145 21(a-i) All 2′-deoxynucleosides  4(a-i) 146 22(a-i) All 2′-deoxynucleosides  4(a-i) 147  1(a-i) All 2′-deoxynucleosides  5(a-i) 148  2(a-i) All 2′-deoxynucleosides  5(a-i) 149  3(a-i) All 2′-deoxynucleosides  5(a-i) 150  4(a-i) All 2′-deoxynucleosides  5(a-i) 151  5(a-i) All 2′-deoxynucleosides  5(a-i) 152  6(a-i) All 2′-deoxynucleosides  5(a-i) 153  7(a-i) All 2′-deoxynucleosides  5(a-i) 154  8(a-i) All 2′-deoxynucleosides  5(a-i) 155  9(a-i) All 2′-deoxynucleosides  5(a-i) 156 10(a-i) All 2′-deoxynucleosides  5(a-i) 157 11(a-i) All 2′-deoxynucleosides  5(a-i) 158 12(a-i) All 2′-deoxynucleosides  5(a-i) 159 13(a-i) All 2′-deoxynucleosides  5(a-i) 160 14(a-i) All 2′-deoxynucleosides  5(a-i) 161 15(a-i) All 2′-deoxynucleosides  5(a-i) 162 16(a-i) All 2′-deoxynucleosides  5(a-i) 163 17(a-i) All 2′-deoxynucleosides  5(a-i) 164 18(a-i) All 2′-deoxynucleosides  5(a-i) 165 19(a-i) All 2′-deoxynucleosides  5(a-i) 166 20(a-i) All 2′-deoxynucleosides  5(a-i) 167 21(a-i) All 2′-deoxynucleosides  5(a-i) 168 22(a-i) All 2′-deoxynucleosides  5(a-i) 169  1(a-i) All 2′-deoxynucleosides  6(a-i) 170  2(a-i) All 2′-deoxynucleosides  6(a-i) 171  3(a-i) All 2′-deoxynucleosides  6(a-i) 172  4(a-i) All 2′-deoxynucleosides  6(a-i) 173  5(a-i) All 2′-deoxynucleosides  6(a-i) 174  6(a-i) All 2′-deoxynucleosides  6(a-i) 175  7(a-i) All 2′-deoxynucleosides  6(a-i) 176  8(a-i) All 2′-deoxynucleosides  6(a-i) 177  9(a-i) All 2′-deoxynucleosides  6(a-i) 178 10(a-i) All 2′-deoxynucleosides  6(a-i) 179 11(a-i) All 2′-deoxynucleosides  6(a-i) 180 12(a-i) All 2′-deoxynucleosides  6(a-i) 181 13(a-i) All 2′-deoxynucleosides  6(a-i) 182 14(a-i) All 2′-deoxynucleosides  6(a-i) 183 15(a-i) All 2′-deoxynucleosides  6(a-i) 184 16(a-i) All 2′-deoxynucleosides  6(a-i) 184 17(a-i) All 2′-deoxynucleosides  6(a-i) 186 18(a-i) All 2′-deoxynucleosides  6(a-i) 187 19(a-i) All 2′-deoxynucleosides  6(a-i) 188 20(a-i) All 2′-deoxynucleosides  6(a-i) 189 21(a-i) All 2′-deoxynucleosides  6(a-i) 190 22(a-i) All 2′-deoxynucleosides  6(a-i) 191  1(a-i) All 2′-deoxynucleosides  7(a-i) 192  2(a-i) All 2′-deoxynucleosides  7(a-i) 193  3(a-i) All 2′-deoxynucleosides  7(a-i) 194  4(a-i) All 2′-deoxynucleosides  7(a-i) 195  5(a-i) All 2′-deoxynucleosides  7(a-i) 196  6(a-i) All 2′-deoxynucleosides  7(a-i) 197  7(a-i) All 2′-deoxynucleosides  7(a-i) 198  8(a-i) All 2′-deoxynucleosides  7(a-i) 199  9(a-i) All 2′-deoxynucleosides  7(a-i) 200 10(a-i) All 2′-deoxynucleosides  7(a-i) 201 11(a-i) All 2′-deoxynucleosides  7(a-i) 202 12(a-i) All 2′-deoxynucleosides  7(a-i) 203 13(a-i) All 2′-deoxynucleosides  7(a-i) 204 14(a-i) All 2′-deoxynucleosides  7(a-i) 205 15(a-i) All 2′-deoxynucleosides  7(a-i) 206 16(a-i) All 2′-deoxynucleosides  7(a-i) 207 17(a-i) All 2′-deoxynucleosides  7(a-i) 208 18(a-i) All 2′-deoxynucleosides  7(a-i) 209 19(a-i) All 2′-deoxynucleosides  7(a-i) 210 20(a-i) All 2′-deoxynucleosides  7(a-i) 211 21(a-i) All 2′-deoxynucleosides  7(a-i) 212 22(a-i) All 2′-deoxynucleosides  7(a-i) 213  1(a-i) All 2′-deoxynucleosides  8(a-i) 214  2(a-i) All 2′-deoxynucleosides  8(a-i) 215  3(a-i) All 2′-deoxynucleosides  8(a-i) 216  4(a-i) All 2′-deoxynucleosides  8(a-i) 217  5(a-i) All 2′-deoxynucleosides  8(a-i) 218  6(a-i) All 2′-deoxynucleosides  8(a-i) 219  7(a-i) All 2′-deoxynucleosides  8(a-i) 220  8(a-i) All 2′-deoxynucleosides  8(a-i) 221  9(a-i) All 2′-deoxynucleosides  8(a-i) 222 10(a-i) All 2′-deoxynucleosides  8(a-i) 223 11(a-i) All 2′-deoxynucleosides  8(a-i) 224 12(a-i) All 2′-deoxynucleosides  8(a-i) 225 13(a-i) All 2′-deoxynucleosides  8(a-i) 226 14(a-i) All 2′-deoxynucleosides  8(a-i) 227 15(a-i) All 2′-deoxynucleosides  8(a-i) 228 16(a-i) All 2′-deoxynucleosides  8(a-i) 229 17(a-i) All 2′-deoxynucleosides  8(a-i) 230 18(a-i) All 2′-deoxynucleosides  8(a-i) 231 19(a-i) All 2′-deoxynucleosides  8(a-i) 232 20(a-i) All 2′-deoxynucleosides  8(a-i) 233 21(a-i) All 2′-deoxynucleosides  8(a-i) 234 22(a-i) All 2′-deoxynucleosides  8(a-i) 235  1(a-i) All 2′-deoxynucleosides  9(a-i) 236  2(a-i) All 2′-deoxynucleosides  9(a-i) 237  3(a-l) All 2′-deoxynucleosides  9(a-i) 238  4(a-i) All 2′-deoxynucleosides  9(a-i) 239  5(a-i) All 2′-deoxynucleosides  9(a-i) 240  6(a-i) All 2′-deoxynucleosides  9(a-i) 241  7(a-i) All 2′-deoxynucleosides  9(a-i) 242  8(a-i) All 2′-deoxynucleosides  9(a-i) 243  9(a-i) All 2′-deoxynucleosides  9(a-i) 244 10(a-i) All 2′-deoxynucleosides  9(a-i) 245 11(a-i) All 2′-deoxynucleosides  9(a-i) 246 12(a-i) All 2′-deoxynucleosides  9(a-i) 247 13(a-i) All 2′-deoxynucleosides  9(a-i) 248 14(a-i) All 2′-deoxynucleosides  9(a-i) 249 15(a-i) All 2′-deoxynucleosides  9(a-i) 250 16(a-i) All 2′-deoxynucleosides  9(a-i) 251 17(a-i) All 2′-deoxynucleosides  9(a-i) 252 18(a-i) All 2′-deoxynucleosides  9(a-i) 253 19(a-i) All 2′-deoxynucleosides  9(a-i) 254 20(a-i) All 2′-deoxynucleosides  9(a-i) 255 21(a-i) All 2′-deoxynucleosides  9(a-i) 256 22(a-i) All 2′-deoxynucleosides  9(a-i) 257  1(a-i) All 2′-deoxynucleosides 10(a-i) 258  2(a-i) All 2′-deoxynucleosides 10(a-i) 259  3(a-i) All 2′-deoxynucleosides 10(a-i) 260  4(a-i) All 2′-deoxynucleosides 10(a-i) 261  5(a-i) All 2′-deoxynucleosides 10(a-i) 262  6(a-i) All 2′-deoxynucleosides 10(a-i) 263  7(a-i) All 2′-deoxynucleosides 10(a-i) 264  8(a-i) All 2′-deoxynucleosides 10(a-i) 265  9(a-i) All 2′-deoxynucleosides 10(a-i) 266 10(a-i) All 2′-deoxynucleosides 10(a-i) 267 11(a-i) All 2′-deoxynucleosides 10(a-i) 268 12(a-i) All 2′-deoxynucleosides 10(a-i) 269 13(a-i) All 2′-deoxynucleosides 10(a-i) 270 14(a-i) All 2′-deoxynucleosides 10(a-i) 271 15(a-i) All 2′-deoxynucleosides 10(a-i) 272 16(a-i) All 2′-deoxynucleosides 10(a-i) 273 17(a-i) All 2′-deoxynucleosides 10(a-i) 274 18(a-i) All 2′-deoxynucleosides 10(a-i) 275 19(a-i) All 2′-deoxynucleosides 10(a-i) 276 20(a-i) All 2′-deoxynucleosides 10(a-i) 277 21(a-i) All 2′-deoxynucleosides 10(a-i) 278 22(a-i) All 2′-deoxynucleosides 10(a-i) 279 1(a)-22(a) All 2′-deoxynucleosides 1(a)-10(a) 280 1(b)-22(b) All 2′-deoxynucleosides 1(a)-10(a) 281 1(c)-22(c) All 2′-deoxynucleosides 1(a)-10(a) 282 1(d)-22(d) All 2′-deoxynucleosides 1(a)-10(a) 283 1(e)-22(e) All 2′-deoxynucleosides 1(a)-10(a) 284 1(f)-22(f) All 2′-deoxynucleosides 1(a)-10(a) 285 1(g)-22(g) All 2′-deoxynucleosides 1(a)-10(a) 286 1(h)-22(h) All 2′-deoxynucleosides 1(a)-10(a) 287 1(i)-22(i) All 2′-deoxynucleosides 1(a)-10(a) 288 1(a)-22(a) All 2′-deoxynucleosides 1(b)-10(b) 289 1(b)-22(b) All 2′-deoxynucleosides 1(b)-10(b) 290 1(c)-22(c) All 2′-deoxynucleosides 1(b)-10(b) 291 1(d)-22(d) All 2′-deoxynucleosides 1(b)-10(b) 292 1(e)-22(e) All 2′-deoxynucleosides 1(b)-10(b) 293 1(f)-22(f) All 2′-deoxynucleosides 1(b)-10(b) 294 1(g)-22(g) All 2′-deoxynucleosides 1(b)-10(b) 295 1(h)-22(h) All 2′-deoxynucleosides 1(b)-10(b) 296 1(i)-22(i) All 2′-deoxynucleosides 1(b)-10(b) 297 1(a)-22(a) All 2′-deoxynucleosides 1(c)-10(c) 298 1(b)-22(b) All 2′-deoxynucleosides 1(c)-10(c) 299 1(c)-22(c) All 2′-deoxynucleosides 1(c)-10(c) 300 1(d)-22(d) All 2′-deoxynucleosides 1(c)-10(c) 301 1(e)-22(e) All 2′-deoxynucleosides 1(c)-10(c) 302 1(f)-22(f) All 2′-deoxynucleosides 1(c)-10(c) 303 1(g)-22(g) All 2′-deoxynucleosides 1(c)-10(c) 304 1(h)-22(h) All 2′-deoxynucleosides 1(c)-10(c) 305 1(i)-22(i) All 2′-deoxynucleosides 1(c)-10(c) 306 1(a)-22(a) All 2′-deoxynucleosides 1(d)-10(d) 307 1(b)-22(b) All 2′-deoxynucleosides 1(d)-10(d) 308 1(c)-22(c) All 2′-deoxynucleosides 1(d)-10(d) 309 1(d)-22(d) All 2′-deoxynucleosides 1(d)-10(d) 310 1(e)-22(e) All 2′-deoxynucleosides 1(d)-10(d) 311 1(f)-22(f) All 2′-deoxynucleosides 1(d)-10(d) 312 1(g)-22(g) All 2′-deoxynucleosides 1(d)-10(d) 313 1(h)-22(h) All 2′-deoxynucleosides 1(d)-10(d) 314 1(i)-22(i) All 2′-deoxynucleosides 1(d)-10(d) 315 1(a)-22(a) All 2′-deoxynucleosides 1(e)-10(e) 316 1(b)-22(b) All 2′-deoxynucleosides 1(e)-10(e) 317 1(c)-22(c) All 2′-deoxynucleosides 1(e)-10(e) 318 1(d)-22(d) All 2′-deoxynucleosides 1(e)-10(e) 319 1(e)-22(e) All 2′-deoxynucleosides 1(e)-10(e) 320 1(f)-22(f) All 2′-deoxynucleosides 1(e)-10(e) 321 1(g)-22(g) All 2′-deoxynucleosides 1(e)-10(e) 322 1(h)-22(h) All 2′-deoxynucleosides 1(e)-10(e) 323 1(i)-22(i) All 2′-deoxynucleosides 1(e)-10(e) 324 1(a)-22(a) All 2′-deoxynucleosides 1(f)-10(f) 325 1(b)-22(b) All 2′-deoxynucleosides 1(f)-10(f) 326 1(c)-22(c) All 2′-deoxynucleosides 1(f)-10(f) 327 1(d)-22(d) All 2′-deoxynucleosides 1(f)-10(f) 328 1(e)-22(e) All 2′-deoxynucleosides 1(f)-10(f) 329 1(f)-22(f) All 2′-deoxynucleosides 1(f)-10(f) 330 1(g)-22(g) All 2′-deoxynucleosides 1(f)-10(f) 331 1(h)-22(h) All 2′-deoxynucleosides 1(f)-10(f) 332 1(i)-22(i) All 2′-deoxynucleosides 1(f)-10(f) 333 1(a)-22(a) All 2′-deoxynucleosides 1(g)-10(g) 334 1(b)-22(b) All 2′-deoxynucleosides 1(g)-10(g) 335 1(e)-22(c) All 2′-deoxynucleosides 1(g)-10(g) 336 1(d)-22(d) All 2′-deoxynucleosides 1(g)-10(g) 337 1(e)-22(e) All 2′-deoxynucleosides 1(g)-10(g) 338 1(f)-22(f) All 2′-deoxynucleosides 1(g)-10(g) 339 1(g)-22(g) All 2′-deoxynucleosides 1(g)-10(g) 340 1(h)-22(h) All 2′-deoxynucleosides 1(g)-10(g) 341 1(i)-22(i) All 2′-deoxynucleosides 1(g)-10(g) 342 1(a)-22(a) All 2′-deoxynucleosides 1(h)-10(h) 343 1(b)-22(b) All 2′-deoxynucleosides 1(h)-10(h) 344 1(c)-22(c) All 2′-deoxynucleosides 1(h)-10(h) 345 1(d)-22(d) All 2′-deoxynucleosides 1(h)-10(h) 346 1(e)-22(e) All 2′-deoxynucleosides 1(h)-10(h) 347 1(f)-22(f) All 2′-deoxynucleosides 1(h)-10(h) 348 1(g)-22(g) All 2′-deoxynucleosides 1(h)-10(h) 349 1(h)-22(h) All 2′-deoxynucleosides 1(h)-10(h) 350 1(i)-22(i) All 2′-deoxynucleosides 1(h)-10(h) 351 1(a)-22(a) All 2′-deoxynucleosides 1(i)-10(i) 352 1(b)-22(b) All 2′-deoxynucleosides 1(i)-10(i) 353 1(c)-22(c) All 2′-deoxynucleosides 1(i)-10(i) 354 1(d)-22(d) All 2′-deoxynucleosides 1(i)-10(i) 355 1(e)-22(e) All 2′-deoxynucleosides 1(i)-10(i) 356 1(f)-22(f) All 2′-deoxynucleosides 1(i)-10(i) 357 1(g)-22(g) All 2′-deoxynucleosides 1(i)-10(i) 358 1(h)-22(h) All 2′-deoxynucleosides 1(i)-10(i) 359 1(i)-22(i) All 2′-deoxynucleosides 1(i)-10(i) 360  1(a-l) All 2′-deoxynucleosides  1(a-l) 361  2(a-l) All 2′-deoxynucleosides  1(a-l) 362  3(a-l) All 2′-deoxynucleosides  1(a-l) 363  4(a-l) All 2′-deoxynucleosides  1(a-l) 364  5(a-l) All 2′-deoxynucleosides  1(a-l) 365  6(a-l) All 2′-deoxynucleosides  1(a-l) 366  7(a-l) All 2′-deoxynucleosides  1(a-l) 367  8(a-l) All 2′-deoxynucleosides  1(a-l) 368  9(a-l) All 2′-deoxynucleosides  1(a-l) 369 10(a-l) All 2′-deoxynucleosides  1(a-l) 370 11(a-l) All 2′-deoxynucleosides  1(a-l) 371 12(a-l) All 2′-deoxynucleosides  1(a-l) 372 13(a-l) All 2′-deoxynucleosides  1(a-l) 373 14(a-l) All 2′-deoxynucleosides  1(a-l) 374 15(a-l) All 2′-deoxynucleosides  1(a-l) 375 16(a-l) All 2′-deoxynucleosides  1(a-l) 376 17(a-l) All 2′-deoxynucleosides  1(a-l) 377 18(a-l) All 2′-deoxynucleosides  1(a-l) 378 19(a-l) All 2′-deoxynucleosides  1(a-l) 379 20(a-l) All 2′-deoxynucleosides  1(a-l) 380 21(a-l) All 2′-deoxynucleosides  1(a-l) 381 22(a-l) All 2′-deoxynucleosides  1(a-l) 382  1(a-l) All 2′-deoxynucleosides  2(a-l) 383  2(a-l) All 2′-deoxynucleosides  2(a-l) 384  3(a-l) All 2′-deoxynucleosides  2(a-l) 385  4(a-l) All 2′-deoxynucleosides  2(a-l) 386  5(a-l) All 2′-deoxynucleosides  2(a-l) 387  6(a-l) All 2′-deoxynucleosides  2(a-l) 388  7(a-l) All 2′-deoxynucleosides  2(a-l) 389  8(a-l) All 2′-deoxynucleosides  2(a-l) 390  9(a-l) All 2′-deoxynucleosides  2(a-l) 391 10(a-l) All 2′-deoxynucleosides  2(a-l) 392 11(a-l) All 2′-deoxynucleosides  2(a-l) 393 12(a-l) All 2′-deoxynucleosides  2(a-l) 394 13(a-l) All 2′-deoxynucleosides  2(a-l) 395 14(a-l) All 2′-deoxynucleosides  2(a-l) 396 15(a-l) All 2′-deoxynucleosides  2(a-l) 397 16(a-l) All 2′-deoxynucleosides  2(a-l) 398 17(a-l) All 2′-deoxynucleosides  2(a-l) 399 18(a-l) All 2′-deoxynucleosides  2(a-l) 400 19(a-l) All 2′-deoxynucleosides  2(a-l) 401 20(a-l) All 2′-deoxynucleosides  2(a-l) 402 21(a-l) All 2′-deoxynucleosides  2(a-l) 403 22(a-l) All 2′-deoxynucleosides  2(a-l) 404  1(a-l) All 2′-deoxynucleosides  3(a-l) 405  2(a-l) All 2′-deoxynucleosides  3(a-l) 406  3(a-l) All 2′-deoxynucleosides  3(a-l) 407  4(a-l) All 2′-deoxynucleosides  3(a-l) 408  5(a-l) All 2′-deoxynucleosides  3(a-l) 409  6(a-l) All 2′-deoxynucleosides  3(a-l) 410  7(a-l) All 2′-deoxynucleosides  3(a-l) 411  8(a-l) All 2′-deoxynucleosides  3(a-l) 412  9(a-l) All 2′-deoxynucleosides  3(a-l) 413 10(a-l) All 2′-deoxynucleosides  3(a-l) 414 11(a-l) All 2′-deoxynucleosides  3(a-l) 415 12(a-l) All 2′-deoxynucleosides  3(a-l) 416 13(a-l) All 2′-deoxynucleosides  3(a-l) 417 14(a-l) All 2′-deoxynucleosides  3(a-l) 418 15(a-l) All 2′-deoxynucleosides  3(a-l) 419 16(a-l) All 2′-deoxynucleosides  3(a-l) 420 17(a-l) All 2′-deoxynucleosides  3(a-l) 421 18(a-l) All 2′-deoxynucleosides  3(a-l) 422 19(a-l) All 2′-deoxynucleosides  3(a-l) 423 20(a-l) All 2′-deoxynucleosides  3(a-l) 424 21(a-l) All 2′-deoxynucleosides  3(a-l) 425 22(a-l) All 2′-deoxynucleosides  3(a-l) 426  1(a-l) All 2′-deoxynucleosides  4(a-l) 427  2(a-l) All 2′-deoxynucleosides  4(a-l) 428  3(a-l) All 2′-deoxynucleosides  4(a-l) 429  4(a-l) All 2′-deoxynucleosides  4(a-l) 430  5(a-l) All 2′-deoxynucleosides  4(a-l) 431  6(a-l) All 2′-deoxynucleosides  4(a-l) 432  7(a-l) All 2′-deoxynucleosides  4(a-l) 433  8(a-l) All 2′-deoxynucleosides  4(a-l) 434  9(a-l) All 2′-deoxynucleosides  4(a-l) 435 10(a-l) All 2′-deoxynucleosides  4(a-l) 436 11(a-l) All 2′-deoxynucleosides  4(a-l) 437 12(a-l) All 2′-deoxynucleosides  4(a-l) 438 13(a-l) All 2′-deoxynucleosides  4(a-l) 439 14(a-l) All 2′-deoxynucleosides  4(a-l) 440 15(a-l) All 2′-deoxynucleosides  4(a-l) 441 16(a-l) All 2′-deoxynucleosides  4(a-l) 442 17(a-l) All 2′-deoxynucleosides  4(a-l) 443 18(a-l) All 2′-deoxynucleosides  4(a-l) 444 19(a-l) All 2′-deoxynucleosides  4(a-l) 445 20(a-l) All 2′-deoxynucleosides  4(a-l) 446 21(a-l) All 2′-deoxynucleosides  4(a-l) 447 22(a-l) All 2′-deoxynucleosides  4(a-l) 448  1(a-l) All 2′-deoxynucleosides  5(a-l) 449  2(a-l) All 2′-deoxynucleosides  5(a-l) 450  3(a-l) All 2′-deoxynucleosides  5(a-l) 451  4(a-l) All 2′-deoxynucleosides  5(a-l) 452  5(a-l) All 2′-deoxynucleosides  5(a-l) 453  6(a-l) All 2′-deoxynucleosides  5(a-l) 454  7(a-l) All 2′-deoxynucleosides  5(a-l) 455  8(a-l) All 2′-deoxynucleosides  5(a-l) 456  9(a-l) All 2′-deoxynucleosides  5(a-l) 457 10(a-l) All 2′-deoxynucleosides  5(a-l) 458 11(a-l) All 2′-deoxynucleosides  5(a-l) 459 12(a-l) All 2′-deoxynucleosides  5(a-l) 460 13(a-l) All 2′-deoxynucleosides  5(a-l) 461 14(a-l) All 2′-deoxynucleosides  5(a-l) 462 15(a-l) All 2′-deoxynucleosides  5(a-l) 463 16(a-l) All 2′-deoxynucleosides  5(a-l) 464 17(a-l) All 2′-deoxynucleosides  5(a-l) 465 18(a-l) All 2′-deoxynucleosides  5(a-l) 466 19(a-l) All 2′-deoxynucleosides  5(a-l) 467 20(a-l) All 2′-deoxynucleosides  5(a-l) 468 21(a-l) All 2′-deoxynucleosides  5(a-l) 469 22(a-l) All 2′-deoxynucleosides  5(a-l) 470  1(a-l) All 2′-deoxynucleosides  6(a-l) 471  2(a-l) All 2′-deoxynucleosides  6(a-l) 472  3(a-l) All 2′-deoxynucleosides  6(a-l) 473  4(a-l) All 2′-deoxynucleosides  6(a-l) 474  5(a-l) All 2′-deoxynucleosides  6(a-l) 475  6(a-l) All 2′-deoxynucleosides  6(a-l) 476  7(a-l) All 2′-deoxynucleosides  6(a-l) 477  8(a-l) All 2′-deoxynucleosides  6(a-l) 478  9(a-l) All 2′-deoxynucleosides  6(a-l) 479 10(a-l) All 2′-deoxynucleosides  6(a-l) 480 11(a-l) All 2′-deoxynucleosides  6(a-l) 481 12(a-l) All 2′-deoxynucleosides  6(a-l) 482 13(a-l) All 2′-deoxynucleosides  6(a-l) 483 14(a-l) All 2′-deoxynucleosides  6(a-l) 484 15(a-l) All 2′-deoxynucleosides  6(a-l) 485 16(a-l) All 2′-deoxynucleosides  6(a-l) 486 17(a-l) All 2′-deoxynucleosides  6(a-l) 487 18(a-l) All 2′-deoxynucleosides  6(a-l) 488 19(a-l) All 2′-deoxynucleosides  6(a-l) 489 20(a-l) All 2′-deoxynucleosides  6(a-l) 490 21(a-l) All 2′-deoxynucleosides  6(a-l) 491 22(a-l) All 2′-deoxynucleosides  6(a-l) 492  1(a-l) All 2′-deoxynucleosides  7(a-l) 493  2(a-l) All 2′-deoxynucleosides  7(a-l) 494  3(a-l) All 2′-deoxynucleosides  7(a-l) 495  4(a-l) All 2′-deoxynucleosides  7(a-l) 496  5(a-l) All 2′-deoxynucleosides  7(a-l) 497  6(a-l) All 2′-deoxynucleosides  7(a-l) 498  7(a-l) All 2′-deoxynucleosides  7(a-l) 499  8(a-l) All 2′-deoxynucleosides  7(a-l) 500  9(a-l) All 2′-deoxynucleosides  7(a-l) 501 10(a-l) All 2′-deoxynucleosides  7(a-l) 502 11(a-l) All 2′-deoxynucleosides  7(a-l) 503 12(a-l) All 2′-deoxynucleosides  7(a-l) 504 13(a-l) All 2′-deoxynucleosides  7(a-l) 505 14(a-l) All 2′-deoxynucleosides  7(a-l) 506 15(a-l) All 2′-deoxynucleosides  7(a-l) 507 16(a-l) All 2′-deoxynucleosides  7(a-l) 508 17(a-l) All 2′-deoxynucleosides  7(a-l) 509 18(a-l) All 2′-deoxynucleosides  7(a-l) 510 19(a-l) All 2′-deoxynucleosides  7(a-l) 511 20(a-l) All 2′-deoxynucleosides  7(a-l) 512 21(a-l) All 2′-deoxynucleosides  7(a-l) 513 22(a-l) All 2′-deoxynucleosides  7(a-l) 514  1(a-l) All 2′-deoxynucleosides  8(a-l) 515  2(a-l) All 2′-deoxynucleosides  8(a-l) 516  3(a-l) All 2′-deoxynucleosides  8(a-l) 517  4(a-l) All 2′-deoxynucleosides  8(a-l) 518  5(a-l) All 2′-deoxynucleosides  8(a-l) 519  6(a-l) All 2′-deoxynucleosides  8(a-l) 520  7(a-l) All 2′-deoxynucleosides  8(a-l) 521  8(a-l) All 2′-deoxynucleosides  8(a-l) 522  9(a-l) All 2′-deoxynucleosides  8(a-l) 523 10(a-l) All 2′-deoxynucleosides  8(a-l) 524 11(a-l) All 2′-deoxynucleosides  8(a-l) 525 12(a-l) All 2′-deoxynucleosides  8(a-l) 526 13(a-l) All 2′-deoxynucleosides  8(a-l) 527 14(a-l) All 2′-deoxynucleosides  8(a-l) 528 15(a-l) All 2′-deoxynucleosides  8(a-l) 529 16(a-l) All 2′-deoxynucleosides  8(a-l) 530 17(a-l) All 2′-deoxynucleosides  8(a-l) 531 18(a-l) All 2′-deoxynucleosides  8(a-l) 532 19(a-l) All 2′-deoxynucleosides  8(a-l) 533 20(a-l) All 2′-deoxynucleosides  8(a-l) 534 21(a-l) All 2′-deoxynucleosides  8(a-l) 535 22(a-l) All 2′-deoxynucleosides  8(a-l) 536  1(a-l) All 2′-deoxynucleosides  9(a-l) 537  2(a-l) All 2′-deoxynucleosides  9(a-l) 538  3(a-l) All 2′-deoxynucleosides  9(a-l) 539  4(a-l) All 2′-deoxynucleosides  9(a-l) 540  5(a-l) All 2′-deoxynucleosides  9(a-l) 541  6(a-l) All 2′-deoxynucleosides  9(a-l) 542  7(a-l) All 2′-deoxynucleosides  9(a-l) 543  8(a-l) All 2′-deoxynucleosides  9(a-l) 544  9(a-l) All 2′-deoxynucleosides  9(a-l) 545 10(a-l) All 2′-deoxynucleosides  9(a-l) 546 11(a-l) All 2′-deoxynucleosides  9(a-l) 547 12(a-l) All 2′-deoxynucleosides  9(a-l) 548 13(a-l) All 2′-deoxynucleosides  9(a-l) 549 14(a-l) All 2′-deoxynucleosides  9(a-l) 550 15(a-l) All 2′-deoxynucleosides  9(a-l) 551 16(a-l) All 2′-deoxynucleosides  9(a-l) 552 17(a-l) All 2′-deoxynucleosides  9(a-l) 553 18(a-l) All 2′-deoxynucleosides  9(a-l) 554 19(a-l) All 2′-deoxynucleosides  9(a-l) 555 20(a-l) All 2′-deoxynucleosides  9(a-l) 556 21(a-l) All 2′-deoxynucleosides  9(a-l) 557 22(a-l) All 2′-deoxynucleosides  9(a-l) 558  1(a-l) All 2′-deoxynucleosides 10(a-l) 559  2(a-l) All 2′-deoxynucleosides 10(a-l) 560  3(a-l) All 2′-deoxynucleosides 10(a-l) 561  4(a-l) All 2′-deoxynucleosides 10(a-l) 562  5(a-l) All 2′-deoxynucleosides 10(a-l) 563  6(a-l) All 2′-deoxynucleosides 10(a-l) 564  7(a-l) All 2′-deoxynucleosides 10(a-l) 565  8(a-l) All 2′-deoxynucleosides 10(a-l) 566  9(a-l) All 2′-deoxynucleosides 10(a-l) 567 10(a-l) All 2′-deoxynucleosides 10(a-l) 568 11(a-l) All 2′-deoxynucleosides 10(a-l) 569 12(a-l) All 2′-deoxynucleosides 10(a-l) 570 13(a-l) All 2′-deoxynucleosides 10(a-l) 571 14(a-l) All 2′-deoxynucleosides 10(a-l) 572 15(a-l) All 2′-deoxynucleosides 10(a-l) 573 16(a-l) All 2′-deoxynucleosides 10(a-l) 574 17(a-l) All 2′-deoxynucleosides 10(a-l) 575 18(a-l) All 2′-deoxynucleosides 10(a-l) 576 19(a-l) All 2′-deoxynucleosides 10(a-l) 577 20(a-l) All 2′-deoxynucleosides 10(a-l) 578 21(a-l) All 2′-deoxynucleosides 10(a-l) 579 22(a-l) All 2′-deoxynucleosides 10(a-l) 580 1(j)-22(j) All 2′-deoxynucleosides 1(a)-10(a) 581 1(k)-22(k) All 2′-deoxynucleosides 1(a)-10(a) 582 1(l)-22(l) All 2′-deoxynucleosides 1(a)-10(a) 583 1(j)-22(j) All 2′-deoxynucleosides 1(b)-10(b) 584 1(k)-22(k) All 2′-deoxynucleosides 1(b)-10(b) 585 1(l)-22(l) All 2′-deoxynucleosides 1(b)-10(b) 586 1(j)-22(j) All 2′-deoxynucleosides 1(c)-10(c) 587 1(k)-22(k) All 2′-deoxynucleosides 1(c)-10(c) 588 1(l)-22(l) All 2′-deoxynucleosides 1(c)-10(c) 589 1(j)-22(j) All 2′-deoxynucleosides 1(d)-10(d) 590 1(k)-22(k) All 2′-deoxynucleosides 1(d)-10(d) 591 1(l)-22(l) All 2′-deoxynucleosides 1(d)-10(d) 592 1(j)-22(j) All 2′-deoxynucleosides 1(e)-10(e) 593 1(k)-22(k) All 2′-deoxynucleosides 1(e)-10(e) 594 1(l)-22(l) All 2′-deoxynucleosides 1(e)-10(e) 595 1(j)-22(j) All 2′-deoxynucleosides 1(f)-10(f) 596 1(k)-22(k) All 2′-deoxynucleosides 1(f)-10(f) 597 1(l)-22(l) All 2′-deoxynucleosides 1(f)-10(f) 598 1(j)-22(j) All 2′-deoxynucleosides 1(g)-10(g) 599 1(k)-22(k) All 2′-deoxynucleosides 1(g)-10(g) 600 1(l)-22(l) All 2′-deoxynucleosides 1(g)-10(g) 601 1(j)-22(j) All 2′-deoxynucleosides 1(h)-10(h) 602 1(k)-22(k) All 2′-deoxynucleosides 1(h)-10(h) 603 1(l)-22(l) All 2′-deoxynucleosides 1(h)-10(h) 604 1(j)-22(j) All 2′-deoxynucleosides 1(i)-10(i) 605 1(k)-22(k) All 2′-deoxynucleosides 1(i)-10(i) 606 1(l)-22(l) All 2′-deoxynucleosides 1(i)-10(i) 607 1(j)-22(j) All 2′-deoxynucleosides 1(j)-10(j) 608 1(k)-22(k) All 2′-deoxynucleosides 1(j)-10(j) 609 1(l)-22(l) All 2′-deoxynucleosides 1(j)-10(j) 610 1(j)-22(j) All 2′-deoxynucleosides 1(k)-10(k) 611 1(k)-22(k) All 2′-deoxynucleosides 1(k)-10(k) 612 1(l)-22(l) All 2′-deoxynucleosides 1(k)-10(k) 612 1(j)-22(j) All 2′-deoxynucleosides 1(l)-10(l) 614 1(k)-22(k) All 2′-deoxynucleosides 1(l)-10(l) 615 1(l)-22(l) All 2′-deoxynucleosides 1(l)-10(l) 616 1k All 2′-deoxynucleosides 1m

In certain embodiments, a gapmer comprises a 5′-wing selected from among the 5′-wings provided herein and any 3′-wing. In certain embodiments, a gapmer comprises a 5′-wing selected from among 1(a-i) to 22(a-i). In certain embodiments, a gapmer comprises a 5′-wing selected from among 1(a-l) to 22(a-l). In certain embodiments, a gapmer comprises a 3′-wing selected from among the 3′-wings provided herein and any 5′-wing. In certain embodiments, a gapmer comprises a 3′-wing selected from among 1(a-i) to 10(a-i). In certain embodiments, a gapmer comprises a 3′-wing selected from among 1(a-l) to 10(a-l).

In certain embodiments, a gapmer has a sugar motif other than: E-K-K-(D)₉-K-K-E; E-E-E-E-K-(D)₉-E-E-E-E-E; E-K-K-K-(D)₉-K-K-K-E; K-E-E-K-(D)₉-K-E-E-K; K-D-D-K-(D)₉-K-D-D-K; K-E-K-E-K-(D)₉-K-E-K-E-K; K-D-K-D-K-(D)₉-K-D-K-D-K; E-K-E-K-(D)₉-K-E-K-E; E-E-E-E-E-K-(D)₈-E-E-E-E-E; or E-K-E-K-E-(D)₉-E-K-E-K-E. In certain embodiments, a gapmer not having one of the above motifs has a sugar motif of Formula I. In certain embodiments, a gapmer not having one of the above motifs has a sugar motif selected from motifs 1-58. In certain embodiments, a gapmer not having one of the above motifs has a sugar motif of Formula I and selected from sugar motifs 1-58. In certain embodiments, a gapmer not having one of the above motifs has a sugar motif of Formula II. In certain embodiments, a gapmer not having one of the above motifs has a sugar motif selected from motifs 1-615. In certain embodiments, a gapmer not having one of the above motifs has a sugar motif of Formula II and selected from sugar motifs 1-615.

In certain embodiments a gapmer comprises a A-(D)₄-A-(D)₄-A-(D)₄-AA motif. In certain embodiments a gapmer comprises a B-(D)₄-A-(D)₄-A-(D)₄-AA motif. In certain embodiments a gapmer comprises a A-(D)₄-B-(D)₄-A-(D)₄-AA motif. In certain embodiments a gapmer comprises a A-(D)₄-A-(D)₄-B-(D)₄-AA motif. In certain embodiments a gapmer comprises a A-(D)₄-A-(D)₄-A-(D)₄-BA motif. In certain embodiments a gapmer comprises a A-(D)₄-A-(D)₄-A-(D)₄-BB motif. In certain embodiments a gapmer comprises a K-(D)₄-K-(D)₄-K-(D)₄-K-E motif.

Certain Internucleoside Linkage Motifs

In certain embodiments, oligonucleotides comprise modified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif. In certain embodiments, internucleoside linkages are arranged in a gapped motif, as described above for sugar modification motif. In such embodiments, the internucleoside linkages in each of two wing regions are different from the internucleoside linkages in the gap region. In certain embodiments the internucleoside linkages in the wings are phosphodiester and the internucleoside linkages in the gap are phosphorothioate. The sugar modification motif is independently selected, so such oligonucleotides having a gapped internucleoside linkage motif may or may not have a gapped sugar modification motif and if it does have a gapped sugar motif, the wing and gap lengths may or may not be the same.

In certain embodiments, oligonucleotides comprise a region having an alternating internucleoside linkage motif. In certain embodiments, oligonucleotides of the present invention comprise a region of uniformly modified internucleoside linkages. In certain such embodiments, the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide is uniformly linked by phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one internucleoside linkage is phosphorothioate.

In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 10 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least block of at least one 12 consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3′ end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3′ end of the oligonucleotide.

Certain Nucleobase Modification Motifs

In certain embodiments, oligonucleotides comprise chemical modifications to nucleobases arranged along the oligonucleotide or region thereof in a defined pattern or nucleobases modification motif. In certain such embodiments, nucleobase modifications are arranged in a gapped motif. In certain embodiments, nucleobase modifications are arranged in an alternating motif. In certain embodiments, each nucleobase is modified. In certain embodiments, none of the nucleobases is chemically modified.

In certain embodiments, oligonucleotides comprise a block of modified nucleobases. In certain such embodiments, the block is at the 3′-end of the oligonucleotide. In certain embodiments the block is within 3 nucleotides of the 3′-end of the oligonucleotide. In certain such embodiments, the block is at the 5′-end of the oligonucleotide. In certain embodiments the block is within 3 nucleotides of the 5′-end of the oligonucleotide.

In certain embodiments, nucleobase modifications are a function of the natural base at a particular position of an oligonucleotide. For example, in certain embodiments each purine or each pyrimidine in an oligonucleotide is modified. In certain embodiments, each adenine is modified. In certain embodiments, each guanine is modified. In certain embodiments, each thymine is modified. In certain embodiments, each cytosine is modified. In certain embodiments, each uracil is modified.

In certain embodiments, some, all, or none of the cytosine moieties in an oligonucleotide are 5-methyl cytosine moieties. Herein, 5-methyl cytosine is not a “modified nucleobase.” Accordingly, unless otherwise indicated, unmodified nucleobases include both cytosine residues having a 5-methyl and those lacking a 5 methyl. In certain embodiments, the methylation state of all or some cytosine nucleobases is specified.

Certain Overall Lengths

In certain embodiments, the present invention provides oligomeric compounds including oligonucleotides of any of a variety of ranges of lengths. In certain embodiments, the invention provides oligomeric compounds or oligonucleotides consisting of X to Y linked nucleosides, where X represents the fewest number of nucleosides in the range and Y represents the largest number of nucleosides in the range. In certain such embodiments, X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X≤Y. For example, in certain embodiments, the invention provides oligomeric compounds which comprise oligonucleotides consisting of 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8 to 13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 19, 8 to 20, 8 to 21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to 27, 8 to 28, 8 to 29, 8 to 30, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to 14, 9 to 15, 9 to 16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21, 9 to 22, 9 to 23, 9 to 24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to 29, 9 to 30, 10 to 11, 10 to 12, 10 to 13, 10 to 14, 10 to 15, 10 to 16, 10 to 17, 10 to 18, 10 to 19, 10 to 20, 10 to 21, 10 to 22, 10 to 23, 10 to 24, 10 to 25, 10 to 26, 10 to 27, 10 to 28, 10 to 29, 10 to 30, 11 to 12, 11 to 13, 11 to 14, 11 to 15, 11 to 16,11 to 17,11 to 18,11 to 19,11 to 20,11 to 21,11 to 22,11 to 23, 11 to 24, 11 to 25, 11 to 26, 11 to 27, 11 to 28, 11 to 29, 11 to 30, 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to 24,12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to 29, 19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to 26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linked nucleosides. In embodiments where the number of nucleosides of an oligomeric compound or oligonucleotide is limited, whether to a range or to a specific number, the oligomeric compound or oligonucleotide may, nonetheless further comprise additional other substituents. For example, an oligonucleotide comprising 8-30 nucleosides excludes oligonucleotides having 31 nucleosides, but, unless otherwise indicated, such an oligonucleotide may further comprise, for example one or more conjugates, terminal groups, or other substituents. In certain embodiments, a gapmer oligonucleotide has any of the above lengths.

In certain embodiments, any of the gapmer motifs provided above, including but not limited to gapmer motifs 1-278 provided in Tables 3 and 4, may have any of the above lengths. One of skill in the art will appreciate that certain lengths may not be possible for certain motifs. For example: a gapmer having a 5′-wing region consisting of four nucleotides, a gap consisting of at least six nucleotides, and a 3′-wing region consisting of three nucleotides cannot have an overall length less than 13 nucleotides. Thus, one would understand that the lower length limit is 13 and that the limit of 10 in “10-20” has no effect in that embodiment.

Further, where an oligonucleotide is described by an overall length range and by regions having specified lengths, and where the sum of specified lengths of the regions is less than the upper limit of the overall length range, the oligonucleotide may have additional nucleosides, beyond those of the specified regions, provided that the total number of nucleosides does not exceed the upper limit of the overall length range. For example, an oligonucleotide consisting of 20-25 linked nucleosides comprising a 5′-wing consisting of 5 linked nucleosides; a 3′-wing consisting of 5 linked nucleosides and a central gap consisting of 10 linked nucleosides (5+5+10=20) may have up to 5 nucleosides that are not part of the 5′-wing, the 3′-wing, or the gap (before reaching the overall length limitation of 25). Such additional nucleosides may be 5′ of the 5′-wing and/or 3′ of the 3′ wing.

Certain Oligonucleotides

In certain embodiments, oligonucleotides of the present invention are characterized by their sugar motif, internucleoside linkage motif, nucleobase modification motif and overall length. In certain embodiments, such parameters are each independent of one another. Thus, each internucleoside linkage of an oligonucleotide having a gapmer sugar motif may be modified or unmodified and may or may not follow the gapmer modification pattern of the sugar modifications. Thus, the internucleoside linkages within the wing regions of a sugar-gapmer may be the same or different from one another and may be the same or different from the internucleoside linkages of the gap region. Likewise, such sugar-gapmer oligonucleotides may comprise one or more modified nucleobase independent of the gapmer pattern of the sugar modifications. One of skill in the art will appreciate that such motifs may be combined to create a variety of oligonucleotides, such as those provided in the non-limiting Table 5 below.

TABLE 10 Certain Oligonucleotides Overall Internucleoside Nucleobase Mod. Length Sugar motif Linkage Motif Motif 12 Gapmer motif selected from 1- uniform PS uniform unmodified 278 14 Gapmer motif selected from 1- 2-14-2 gapmer: PO in uniform unmodified 278 wings and PS in gap 14 Gapmer motif selected from 1- uniform PS uniform unmodified; 278 all C's are 5-meC 16 Gapmer of Formula I uniform PS uniform unmodified; no Cs are 5-meC) 16 Gapmer of Formula I uniform PS uniform unmodified; at least one nucleobase is a 5-meC 16 Gapmer of Formula I and having uniform PS uniform unmodified motif selected from 1-58 17 Gapmer of Formula I and having uniform PO uniform unmodified motif selected from 1-58 17 Gapmer motif selected from 1- uniform PS uniform unmodified 278 17 Gapmer of Formula I uniform PS uniform unmodified 18 Gapmer of Formula I and having uniform PS uniform unmodified motif selected from 1-58 18 Gapmer motif selected from 1- uniform PS uniform unmodified 278 20 Gapmer of Formula I uniform PS uniform unmodified 12 Gapmer motif selected from 1- uniform PS uniform unmodified 359 14 Gapmer motif selected from 1- 2-14-2 gapmer: PO in uniform unmodified 359 wings and PS in gap 14 Gapmer motif selected from 1- uniform PS uniform unmodified; 359 all C's are 5-meC 16 Gapmer of Formula II uniform PS uniform unmodified; no Cs are 5-meC) 16 Gapmer of Formula II uniform PS uniform unmodified; at least one nucleobase is a 5-meC 16 Gapmer of Formula II and having uniform PS uniform unmodified motif selected from 1-359 17 Gapmer of Formula II and having uniform PO uniform unmodified motif selected from 1-359 17 Gapmer motif selected from 1- uniform PS uniform unmodified 359 17 Gapmer of Formula II uniform PS uniform unmodified 18 Gapmer of Formula I and having uniform PS uniform unmodified motif selected from 1-359 18 Gapmer motif selected from 1- uniform PS uniform unmodified 359 20 Gapmer of Formula II uniform PS uniform unmodified 12 Gapmer motif selected from 1- uniform PS uniform unmodified 615 14 Gapmer motif selected from 1- 2-14-2 gapmer: PO in uniform unmodified 615 wings and PS in gap 14 Gapmer motif selected from 1- uniform PS uniform unmodified; 615 all C's are 5-meC 16 Gapmer of Formula I and having uniform PS uniform unmodified motif selected from 1-615 17 Gapmer of Formula I and having uniform PO uniform unmodified motif selected from 1-615 17 Gapmer motif selected from 1- uniform PS uniform unmodified 615 18 Gapmer of Formula land having uniform PS uniform unmodified motif selected from 1-615 18 Gapmer motif selected from 1- uniform PS uniform unmodified 615 The above table is intended only to illustrate and not to limit the various combinations of the parameters of oligonucleotides of the present invention. Herein if a description of an oligonucleotide or oligomeric compound is silent with respect to one or more parameter, such parameter is not limited. Thus, an oligomeric compound described only as having a gapmer sugar motif without further description may have any length, internucleoside linkage motif, and nucleobase modification motif. Unless otherwise indicated, all chemical modifications are independent of nucleobase sequence.

Certain Conjugate Groups

In certain embodiments, oligomeric compounds are modified by attachment of one or more conjugate groups. In general, conjugate groups modify one or more properties of the attached oligomeric compound including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, cellular distribution, cellular uptake, charge and clearance. Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional conjugate linking moiety or conjugate linking group to a parent compound such as an oligomeric compound, such as an oligonucleotide. Conjugate groups includes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes. Certain conjugate groups have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).

In certain embodiments, a conjugate group comprises an active drug substance, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.

In certain embodiments, conjugate groups are directly attached to oligonucleotides in oligomeric compounds. In certain embodiments, conjugate groups are attached to oligonucleotides by a conjugate linking group. In certain such embodiments, conjugate linking groups, including, but not limited to, bifunctional linking moieties such as those known in the art are amenable to the compounds provided herein. Conjugate linking groups are useful for attachment of conjugate groups, such as chemical stabilizing groups, functional groups, reporter groups and other groups to selective sites in a parent compound such as for example an oligomeric compound. In general a bifunctional linking moiety comprises a hydrocarbyl moiety having two functional groups. One of the functional groups is selected to bind to a parent molecule or compound of interest and the other is selected to bind essentially any selected group such as chemical functional group or a conjugate group. In some embodiments, the conjugate linker comprises a chain structure or an oligomer of repeating units such as ethylene glycol or amino acid units. Examples of functional groups that are routinely used in a bifunctional linking moiety include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In some embodiments, bifunctional linking moieties include amino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double or triple bonds), and the like.

Some nonlimiting examples of conjugate linking moieties include pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and 6-aminohexanoic acid (AHEX or AHA). Other linking groups include, but are not limited to, substituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl or substituted or unsubstituted C₂-C₁₀ alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

Conjugate groups may be attached to either or both ends of an oligonucleotide (terminal conjugate groups) and/or at any internal position.

In certain embodiments, conjugate groups are at the 3′-end of an oligonucleotide of an oligomeric compound. In certain embodiments, conjugate groups are near the 3′-end. In certain embodiments, conjugates are attached at the 3′end of an oligomeric compound, but before one or more terminal group nucleosides. In certain embodiments, conjugate groups are placed within a terminal group.

In certain embodiments, the present invention provides oligomeric compounds. In certain embodiments, oligomeric compounds comprise an oligonucleotide. In certain embodiments, an oligomeric compound comprises an oligonucleotide and one or more conjugate and/or terminal groups. Such conjugate and/or terminal groups may be added to oligonucleotides having any of the chemical motifs discussed above. Thus, for example, an oligomeric compound comprising an oligonucleotide having region of alternating nucleosides may comprise a terminal group.

Antisense Compounds

In certain embodiments, oligomeric compounds of the present invention are antisense compounds. Such antisense compounds are capable of hybridizing to a target nucleic acid, resulting in at least one antisense activity. In certain embodiments, antisense compounds specifically hybridize to one or more target nucleic acid. In certain embodiments, a specifically hybridizing antisense compound has a nucleobase sequence comprising a region having sufficient complementarity to a target nucleic acid to allow hybridization and result in antisense activity and insufficient complementarity to any non-target so as to avoid non-specific hybridization to any non-target nucleic acid sequences under conditions in which specific hybridization is desired (e.g., under physiological conditions for in vivo or therapeutic uses, and under conditions in which assays are performed in the case of in vitro assays).

In certain embodiments, the present invention provides antisense compounds comprising oligonucleotides that are fully complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain embodiments, oligonucleotides are 99% complementary to the target nucleic acid. In certain embodiments, oligonucleotides are 95% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 90% complementary to the target nucleic acid.

In certain embodiments, such oligonucleotides are 85% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 80% complementary to the target nucleic acid. In certain embodiments, an antisense compound comprises a region that is fully complementary to a target nucleic acid and is at least 80% complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain such embodiments, the region of full complementarity is from 6 to 14 nucleobases in length.

Certain Antisense Activities and Mechanisms

In certain antisense activities, hybridization of an antisense compound results in recruitment of a protein that cleaves of the target nucleic acid. For example, certain antisense compounds result in RNase H mediated cleavage of target nucleic acid. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. The “DNA” in such an RNA:DNA duplex, need not be unmodified DNA. In certain embodiments, the invention provides antisense compounds that are sufficiently “DNA-like” to elicit RNase H activity. Such DNA-like antisense compounds include, but are not limited to gapmers having unmodified deoxyfuronose sugar moieties in the nucleosides of the gap and modified sugar moieties in the nucleosides of the wings.

Antisense activities may be observed directly or indirectly. In certain embodiments, observation or detection of an antisense activity involves observation or detection of a change in an amount of a target nucleic acid or protein encoded by such target nucleic acid; a change in the ratio of splice variants of a nucleic acid or protein; and/or a phenotypic change in a cell or animal.

In certain embodiments, compounds comprising oligonucleotides having a gapmer motif described herein have desirable properties compared to non-gapmer oligonucleotides or to gapmers having other motifs. In certain circumstances, it is desirable to identify motifs resulting in a favorable combination of potent antisense activity and relatively low toxicity. In certain embodiments, compounds of the present invention have a favorable therapeutic index (measure of potency divided by measure of toxicity).

Certain Target Nucleic Acids

In certain embodiments, antisense compounds comprise or consist of an oligonucleotide comprising a region that is complementary to a target nucleic acid. In certain embodiments, the target nucleic acid is an endogenous RNA molecule. In certain embodiments, the target nucleic acid is a non-coding RNA. In certain such embodiments, the target non-coding RNA is selected from: a long-non-coding RNA, a short non-coding RNA, an intronic RNA molecule, a snoRNA, a scaRNA, a microRNA (including pre-microRNA and mature microRNA), a ribosomal RNA, and promoter directed RNA. In certain embodiments, the target nucleic acid encodes a protein. In certain such embodiments, the target nucleic acid is selected from: an mRNA and a pre-mRNA, including intronic, exonic and untranslated regions. In certain embodiments, oligomeric compounds are at least partially complementary to more than one target nucleic acid. For example, antisense compounds of the present invention may mimic microRNAs, which typically bind to multiple targets.

In certain embodiments, the target nucleic acid is a nucleic acid other than a mature mRNA. In certain embodiments, the target nucleic acid is a nucleic acid other than a mature mRNA or a microRNA. In certain embodiments, the target nucleic acid is a non-coding RNA other than a microRNA. In certain embodiments, the target nucleic acid is a non-coding RNA other than a microRNA or an intronic region of a pre-mRNA. In certain embodiments, the target nucleic acid is a long non-coding RNA. In certain embodiments, the target RNA is an mRNA. In certain embodiments, the target nucleic acid is a pre-mRNA. In certain such embodiments, the target region is entirely within an intron. In certain embodiments, the target region spans an intron/exon junction. In certain embodiments, the target region is at least 50% within an intron. In certain embodiments, the target nucleic acid is selected from among non-coding RNA, including exonic regions of pre-mRNA. In certain embodiments, the target nucleic acid is a ribosomal RNA (rRNA). In certain embodiments, the target nucleic acid is a non-coding RNA associated with splicing of other pre-mRNAs. In certain embodiments, the target nucleic acid is a nuclear-retained non-coding RNA.

In certain embodiments, antisense compounds described herein are complementary to a target nucleic acid comprising a single-nucleotide polymorphism. In certain such embodiments, the antisense compound is capable of modulating expression of one allele of the single-nucleotide polymorphism-containing-target nucleic acid to a greater or lesser extent than it modulates another allele. In certain embodiments an antisense compound hybridizes to a single-nucleotide polymorphism-containing-target nucleic acid at the single-nucleotide polymorphism site. In certain embodiments an antisense compound hybridizes to a single-nucleotide polymorphism-containing-target nucleic acid near the single-nucleotide polymorphism site. In certain embodiments, the target nucleic acid is a Huntingtin gene transcript. In certain embodiments, the target nucleic acid is a single-nucleotide polymorphism-containing-target nucleic acid other than a Huntingtin gene transcript. In certain embodiments, the target nucleic acid is any nucleic acid other than a Huntingtin gene transcript.

Certain Pharmaceutical Compositions

In certain embodiments, the present invention provides pharmaceutical compositions comprising one or more antisense compound. In certain embodiments, such pharmaceutical composition comprises a suitable pharmaceutically acceptable diluent or carrier. In certain embodiments, a pharmaceutical composition comprises a sterile saline solution and one or more antisense compound. In certain embodiments, such pharmaceutical composition consists of a sterile saline solution and one or more antisense compound. In certain embodiments, the sterile saline is pharmaceutical grade saline. In certain embodiments, a pharmaceutical composition comprises one or more antisense compound and sterile water. In certain embodiments, a pharmaceutical composition consists of one or more antisense compound and sterile water. In certain embodiments, the sterile saline is pharmaceutical grade water. In certain embodiments, a pharmaceutical composition comprises one or more antisense compound and phosphate-buffered saline (PBS). In certain embodiments, a pharmaceutical composition consists of one or more antisense compound and sterile phosphate-buffered saline (PBS). In certain embodiments, the sterile saline is pharmaceutical grade PBS.

In certain embodiments, antisense compounds may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

Pharmaceutical compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters. In certain embodiments, pharmaceutical compositions comprising antisense compounds comprise one or more oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at one or both ends of an oligomeric compound which are cleaved by endogenous nucleases within the body, to form the active antisense oligomeric compound.

Lipid moieties have been used in nucleic acid therapies in a variety of methods. In certain such methods, the nucleic acid is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In certain methods, DNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.

In certain embodiments, pharmaceutical compositions provided herein comprise one or more modified oligonucleotides and one or more excipients. In certain such embodiments, excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.

In certain embodiments, a pharmaceutical composition provided herein comprises a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.

In certain embodiments, a pharmaceutical composition provided herein comprises one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present invention to specific tissues or cell types. For example, in certain embodiments, pharmaceutical compositions include liposomes coated with a tissue-specific antibody.

In certain embodiments, a pharmaceutical composition provided herein comprises a co-solvent system. Certain of such co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/v polyethylene glycol 300. The proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80™; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.

In certain embodiments, a pharmaceutical composition provided herein is prepared for oral administration. In certain embodiments, pharmaceutical compositions are prepared for buccal administration.

In certain embodiments, a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, etc.). In certain of such embodiments, a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, such suspensions may also contain suitable stabilizers or agents that increase the solubility of the pharmaceutical agents to allow for the preparation of highly concentrated solutions.

In certain embodiments, a pharmaceutical composition is prepared for transmucosal administration. In certain of such embodiments penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

In certain embodiments, a pharmaceutical composition provided herein comprises an oligonucleotide in a therapeutically effective amount. In certain embodiments, the therapeutically effective amount is sufficient to prevent, alleviate or ameliorate symptoms of a disease or to prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.

In certain embodiments, one or more modified oligonucleotide provided herein is formulated as a prodrug. In certain embodiments, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically more active form of an oligonucleotide. In certain embodiments, prodrugs are useful because they are easier to administer than the corresponding active form. For example, in certain instances, a prodrug may be more bioavailable (e.g., through oral administration) than is the corresponding active form. In certain instances, a prodrug may have improved solubility compared to the corresponding active form. In certain embodiments, prodrugs are less water soluble than the corresponding active form. In certain instances, such prodrugs possess superior transmittal across cell membranes, where water solubility is detrimental to mobility. In certain embodiments, a prodrug is an ester. In certain such embodiments, the ester is metabolically hydrolyzed to carboxylic acid upon administration. In certain instances the carboxylic acid containing compound is the corresponding active form. In certain embodiments, a prodrug comprises a short peptide (polyaminoacid) bound to an acid group. In certain of such embodiments, the peptide is cleaved upon administration to form the corresponding active form.

In certain embodiments, the present invention provides compositions and methods for reducing the amount or activity of a target nucleic acid in a cell. In certain embodiments, the cell is in an animal. In certain embodiments, the animal is a mammal. In certain embodiments, the animal is a rodent. In certain embodiments, the animal is a primate. In certain embodiments, the animal is a non-human primate. In certain embodiments, the animal is a human.

In certain embodiments, the present invention provides methods of administering a pharmaceutical composition comprising an oligomeric compound of the present invention to an animal. Suitable administration routes include, but are not limited to, oral, rectal, transmucosal, intestinal, enteral, topical, suppository, through inhalation, intrathecal, intracerebroventricular, intraperitoneal, intranasal, intraocular, intratumoral, and parenteral (e.g., intravenous, intramuscular, intramedullary, and subcutaneous). In certain embodiments, pharmaceutical intrathecals are administered to achieve local rather than systemic exposures.

Nonlimiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references, GenBank accession numbers, and the like recited in the present application is incorporated herein by reference in its entirety.

Although the sequence listing accompanying this filing identifies each sequence as either “RNA” or “DNA” as required, in reality, those sequences may be modified with any combination of chemical modifications. One of skill in the art will readily appreciate that such designation as “RNA” or “DNA” to describe modified oligonucleotides is, in certain instances, arbitrary. For example, an oligonucleotide comprising a nucleoside comprising a 2′-OH sugar moiety and a thymine base could be described as a DNA having a modified sugar (2′-OH for the natural 2′-H of DNA) or as an RNA having a modified base (thymine (methylated uracil) for natural uracil of RNA).

Accordingly, nucleic acid sequences provided herein, including, but not limited to those in the sequence listing, are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases. By way of further example and without limitation, an oligomeric compound having the nucleobase sequence “ATCGATCG” encompasses any oligomeric compounds having such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence “AUCGAUCG” and those having some DNA bases and some RNA bases such as “AUCGATCG” and oligomeric compounds having other modified or naturally occurring bases, such as “AT^(me)CGAUCG,” wherein ^(me)C indicates a cytosine base comprising a methyl group at the 5-position.

EXAMPLES

The following examples illustrate certain embodiments of the present invention and are not limiting. Moreover, where specific embodiments are provided, the inventors have contemplated generic application of those specific embodiments. For example, disclosure of an oligonucleotide having a particular motif provides reasonable support for additional oligonucleotides having the same or similar motif And, for example, where a particular high-affinity modification appears at a particular position, other high-affinity modifications at the same position are considered suitable, unless otherwise indicated.

Where nucleobase sequences are not provided, to allow assessment of the relative effects of nucleobase sequence and chemical modification, throughout the examples, oligomeric compounds are assigned a “Sequence Code.” Oligomeric compounds having the same Sequence Code have the same nucleobase sequence. Oligomeric compounds having different Sequence Codes have different nucleobase sequences.

Example 1 Modified Antisense Oligonucleotides Targeting Human Target-X

Antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. ISIS 407939, which was described in an earlier publication (WO 2009/061851) was also tested.

The newly designed chimeric antisense oligonucleotides and their motifs are described in Table 11. The internucleoside linkages throughout each gapmer are phosphorothioate linkages (P═S). Nucleosides followed by “d” indicate 2′-deoxyribonucleosides. Nucleosides followed by “k” indicate constrained ethyl (cEt) nucleosides. Nucleosides followed by “e” indicate 2′-O-methythoxylethyl (2′-MOE) nucleosides. “N” indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).

Each gapmer listed in Table 11 is targeted to the human Target-X genomic sequence.

Activity of the newly designed gapmers was compared to a 5-10-5 2′-MOE gapmer, ISIS 407939 targeting human Target-X and is further described in USPN XXX, incorporated herein by reference. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells, and indicate that several of the newly designed antisense oligonucleotides are more potent than ISIS 407939. A total of 771 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 11. Each of the newly designed antisense oligonucleotides provided in Table 10 achieved greater than 80% inhibition and, therefore, are more active than ISIS 407939.

TABLE 11 Inhibition of human Target-X mRNA levels by chimeric antisense oligonucleotides targeted to Target-X ISIS % in- Gap Wing Chemistry SEQ SEQ Sequence (5' to 3') NO hibition Motif Chemistry 5' 3' CODE ID NO NkNkNkNdNdNdNdNkNd 473359  92 3-10-3 Deoxy/ kkk eee 21 19 NdNdNdNdNeNeNe cEt NkNkNkNdNdNdNdNkNd 473360  96 3-10-3 Deoxy/ kkk eee 22 19 NdNdNdNdNeNeNe cEt NkNkNkNdNdNdNdNdNd 473168  94 3-10-3 Full deoxy kkk kkk 23 19 NdNdNdNdNkNkNk NkNkNkNdNdNdNdNdNd 473317  95 3-10-3 Full deoxy kkk eee 23 19 NdNdNdNdNeNeNe NkNkNkNdNdNdNdNkNd 473471  90 3-10-3 Deoxy/ kkk eee 23 19 NdNdNdNdNeNeNe cEt NkNdNkNdNdNdNdNdNd 473620  94 5-9-2 Full deoxy kdkdk ee 23 19 NdNdNdNdNdNeNe NkNkNdNdNdNdNdNdNd 473019  88 2-10-2 Full deoxy kk kk 24 20 NdNdNdNkNk NkNkNdNdNdNdNdNdNd 473020  93 2-10-2 Full deoxy kk kk 25 20 NdNdNdNkNk NkNkNkNdNdNdNdNdNd 473321  93 3-10-3 Full deoxy kkk eee 26 19 NdNdNdNdNeNeNe NkNkNkNdNdNdNdNdNd 473322  94 3-10-3 Full deoxy kkk eee 27 19 NdNdNdNdNeNeNe NkNkNkNdNdNdNdNdNd 473323  96 3-10-3 Full deoxy kkk eee 28 19 NdNdNdNdNeNeNe NkNkNkNdNdNdNdNdNd 473326  94 3-10-3 Full deoxy kkk eee 29 19 NdNdNdNdNeNeNe NkNkNkNdNdNdNdNkNd 473480  92 3-10-3 Deoxy/ kkk eee 29 19 NdNdNdNdNeNeNe cEt NkNkNkNdNdNdNdNdNd 473178  96 3-10-3 Full deoxy kkk kkk 30 19 NdNdNdNdNkNkNk NkNkNkNdNdNdNdNdNd 473327  96 3-10-3 Full deoxy kkk eee 30 19 NdNdNdNdNeNeNe NkNkNkNdNdNdNdNkNd 473481  93 3-10-3 Deoxy/ kkk eee 30 19 NdNdNdNdNeNeNe cEt NkNdNkNdNkNdNdNdNd 473630  89 5-9-2 Full deoxy kdkdk ee 30 19 NdNdNdNdNdNeNe NkNkNdNdNdNdNdNdNd 473029  96 2-10-2 Full deoxy kk kk 31 20 NdNdNdNkNk NkNkNdNdNdNdNdNdNd 472925  93 2-10-2 Full deoxy kk kk 32 20 NdNdNdNkNk NkNkNdNdNdNdNdNdNd 472926  85 2-10-2 Full deoxy kk kk 33 20 NdNdNdNkNk NkNkNkNdNdNdNdNdNd 473195  97 3-10-3 Full deoxy kkk kkk 34 19 NdNdNdNdNkNkNk NkNkNdNdNdNdNdNdNd 473046  90 2-10-2 Full deoxy kk kk 35 20 NdNdNdNkNk NkNkNdNdNdNdNdNdNd 472935  92 2-10-2 Full deoxy kk kk 36 20 NdNdNdNkNk NkNkNkNdNdNdNdNdNd 473089  95 3-10-3 Full deoxy kkk kkk 37 19 NdNdNdNdNkNkNk NkNkNkNdNdNdNdNdNd 473350  93 3-10-3 Full deoxy kkk eee 38 19 NdNdNdNdNeNeNe NkNkNkNdNdNdNdNdNd 473353  93 3-10-3 Full deoxy kkk eee 39 19 NdNdNdNdNeNeNe NkNkNdNdNdNdNdNdNd 473055  91 2-10-2 Full deoxy kk kk 40 20 NdNdNdNkNk NkNkNkNdNdNdNdNkNd 473392  95 3-10-3 Deoxy/ kkk eee 41 19 NdNdNdNdNeNeNe cEt NkNkNkNdNdNdNdNdNd 473095 100 3-10-3 Full deoxy kkk kkk 42 19 NdNdNdNdNkNkNk NkNkNkNdNdNdNdNdNd 473244  99 3-10-3 Full deoxy kkk eee 42 19 NdNdNdNdNeNeNe NkNkNkNdNdNdNdNkNd 473393  99 3-10-3 Deoxy/ kkk eee 42 19 NdNdNdNdNeNeNe cEt NkNdNkNdNkNdNdNdNd 473547  98 5-9-2 Full deoxy kdkdk ee 42 19 NdNdNdNdNdNeNe NkNkNdNdNdNdNdNdNd 472942  87 2-10-2 Full deoxy kk kk 43 20 NdNdNdNkNk NkNkNkNdNdNdNdNdNd 473098  97 3-10-3 Full deoxy kkk kkk 44 19 NdNdNdNdNkNkNk NkNkNkNdNdNdNdNkNd 473408  92 3-10-3 Deoxy/ kkk eee 45 19 NdNdNdNdNeNeNe cEt NkNkNdNdNdNdNdNdNd 472958  89 2-10-2 Full deoxy kk kk 46 20 NdNdNdNkNk NkNkNdNdNdNdNdNdNd 472959  90 2-10-2 Full deoxy kk kk 47 20 NdNdNdNkNk NkNdNkNdNkNdNdNdNd 473566  94 5-9-2 Full deoxy kdkdk ee 48 19 NdNdNdNdNdNeNe NkNdNkNdNkNdNdNdNd 473567  95 5-9-2 Full deoxy kdkdk ee 49 19 NdNdNdNdNdNeNe NkNdNkNdNkNdNdNdNd 473569  92 5-9-2 Full deoxy kdkdk ee 50 19 NdNdNdNdNdNeNe NkNkNdNdNdNdNdNdNd 457851  90 2-10-2 Full deoxy kk kk 51 20 NdNdNdNkNk NkNkNdNdNdNdNdNdNd 472970  91 2-10-2 Full deoxy kk kk 32 20 NdNdNdNkNk NkNkNkNdNdNdNdNdNd 473125  90 3-10-3 Full deoxy kkk kkk 53 19 NdNdNdNdNkNkNk NkNkNkNdNdNdNdNdNd 473274  98 3-10-3 Full deoxy kkk eee 53 19 NdNdNdNdNeNeNe NkNkNkNdNdNdNdNkNd 473428  90 3-10-3 Deoxy/ kkk eee 53 19 NdNdNdNdNeNeNe cEt NkNdNkNdNkNdNdNdNd 473577  93 5-9-2 Full deoxy kdkdk ee 53 19 NdNdNdNdNdNeNe NkNkNdNdNdNdNdNdNd 472976  97 2-10-2 Full deoxy kk kk 54 20 NdNdNdNkNk NkNkNdNdNdNdNdNd 472983  94 2-10-2 Full deoxy kk kk 55 20 NdNdNdNdNkNk NkNkNdNdNdNdNdNd 472984  90 2-10-2 Full deoxy kk kk 56 20 NdNdNdNdNkNk NkNkNkNdNdNdNdNd 473135  97 3-10-3 Full deoxy kkk kkk 57 19 NdNdNdNdNdNkNkNk NkNkNdNdNdNdNdNd 472986  95 2-10-2 Full deoxy kk kk 58 20 NdNdNdNdNkNk NkNkNkNdNdNdNdNd 473137  95 3-10-3 Full deoxy kkk kkk 59 19 NdNdNdNdNdNkNkNk NkNkNkNdNdNdNdNd 473286  95 3-10-3 Full deoxy kkk eee 59 19 NdNdNdNdNdNeNeNe NkNkNkNdNdNdNdNkNd 473440  88 3-10-3 Deoxy/ kkk eee 59 19 NdNdNdNdNeNeNe cEt NkNdNkNdNkNdNdNd 473589  97 5-9-2 Full deoxy kdkdk ee 59 19 NdNdNdNdNdNdNeNe NkNkNdNdNdNdNdNd 472988  85 2-10-2 Full deoxy kk kk 60 20 NdNdNdNdNkNk NkNkNkNdNdNdNdNd 473140  96 3-10-3 Full deoxy kkk kkk 61 19 NdNdNdNdNdNkNkNk NkNkNdNdNdNdNdNd 472991  90 2-10-2 Full deoxy kk kk 62 20 NdNdNdNdNkNk NkNkNkNdNdNdNdNkNd 473444  94 3-10-3 Deoxy/ kkk eee 63 19 NdNdNdNdNeNeNe cEt NkNkNkNdNdNdNdNd 473142  96 3-10-3 Full deoxy kkk kkk 64 19 NdNdNdNdNdNkNkNk NkNkNkNdNdNdNdNd 473291  95 3-10-3 Full deoxy kkk eee 64 19 NdNdNdNdNdNeNeNe NkNdNkNdNkNdNdNd 473594  95 5-9-2 Full deoxy kdkdk ee 64 19 NdNdNdNdNdNdNeNe NkNkNkNdNdNdNdNdNd 473143  97 3-10-3 Full deoxy kkk kkk 65 19 NdNdNdNdNkNkNk NkNkNkNdNdNdNdNd 473292  96 3-10-3 Full deoxy kkk eee 65 19 NdNdNdNdNdNeNeNe NkNkNkNdNdNdNdNkNd 473446  96 3-10-3 Deoxy/ kkk eee 65 19 NdNdNdNdNeNeNe cEt NkNdNkNdNkNdNdNdNd 473595  84 5-9-2 Full deoxy kdkdk ee 65 19 NdNdNdNdNdNeNe NkNkNdNdNdNdNdNdNd 472994  96 2-10-2 Full deoxy kk kk 66 20 NdNdNdNkNk NkNkNkNdNdNdNdNdNd 473144  98 3-10-3 Full deoxy kkk kkk 67 19 NdNdNdNdNkNkNk NkNkNkNdNdNdNdNdNd 473293  96 3-10-3 Full deoxy kkk eee 67 19 NdNdNdNdNeNeNe NkNkNdNdNdNdNdNdNd 472995  96 2-10-2 Full deoxy kk kk 68 20 NdNdNdNkNk NkNkNkNdNdNdNdNd 473294  91 3-10-3 Full deoxy kkk eee 69 19 NdNdNdNdNdNeNeNe NkNdNkNdNkNdNdNdNd 473597  94 5-9-2 Full deoxy kdkdk ee 69 19 NdNdNdNdNdNeNe NkNkNdNdNdNdNdNdNd 472996  94 2-10-2 Full deoxy kk kk 70 20 NdNdNdNkNk NkNkNkNdNdNdNdNd 473295  92 3-10-3 Full deoxy kkk eee 71 19 NdNdNdNdNdNeNeNe NeNeNeNeNeNdNdNdNdNd 407939  80 5-10-5 Full deoxy eeeee eeee 72 21 NdNdNdNdNdNeNeNeNeNe e NkNkNkNdNdNdNdNdNd 473296  98 3-10-3 Full deoxy kkk eee 73 19 NdNdNdNdNeNeNe NkNkNkNdNdNdNdNkNd 473450  95 3-10-3 Deoxy/ kkk eee 73 19 NdNdNdNdNeNeNe cEt NkNkNdNdNdNdNdNdNd 472998  97 2-10-2 Full deoxy kk kk 74 20 NdNdNdNkNk e = 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside

Example 2 Modified Antisense Oligonucleotides Comprising Constrained Ethyl (cEt) and F-HNA Modifications Targeting Human Target-X

Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. ISIS 407939 was also tested.

The newly designed chimeric antisense oligonucleotides and their motifs are described in Table 12. The internucleoside linkages throughout each gapmer are phosphorothioate linkages (P═S). Nucleosides followed by “d” indicate 2′-deoxyribonucleosides. Nucleosides followed by “k” indicate constrained ethyl (cEt) nucleosides. Nucleosides followed by “e” indicate 2′-O-methythoxylethyl (2′-MOE) modified nucleosides. Nucleosides followed by ‘g’ indicate F-HNA modified nucleosides. “N” indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).

Each gapmer listed in Table 12 is targeted to the human Target-X genomic sequence.

Activity of the newly designed gapmers was compared to a 5-10-5 2′-MOE gapmer, ISIS 407939 targeting human Target-X. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells, and demonstrate that several of the newly designed gapmers are more potent than ISIS 407939. A total of 765 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 12. All but one of the newly designed antisense oligonucleotides provided in Table 12 achieved greater than 30% inhibition and, therefore, are more active than ISIS 407939.

TABLE 12 Inhibition of human Target-X mRNA levels by chimeric antisense oligonucleotides targeted to Target-X ISIS % in- Gap Wing Chemistry SEQ SEQ Sequence (5′ to 3′) No hibition Motif Chemistry 5′ 3′ CODE ID NO NgNgNdNdNdNdNdNdNd 482838 81 2-10-2 Full deoxy gg gg 25 20 NdNdNdNgNg NgNgNgNdNdNdNdNdNd 482992 93 3-10-3 Full deoxy ggg ggg 28 19 NdNdNdNdNgNgNg NgNgNgNdNdNdNdNdNd 482996 97 3-10-3 Full deoxy ggg ggg 30 19 NdNdNdNdNgNgNg NgNdNgNdNgNdNdNdNd 483284 82 5-9-2 Full deoxy gdgdg ee 23 19 NdNdNdNdNdNeNe NgNdNgNdNgNdNdNdNd 483289 70 5-9-2 Full deoxy gdgdg ee 27 19 NdNdNdNdNdNeNe NgNdNgNdNgNdNdNdNd 483290 80 5-9-2 Full deoxy gdgdg ee 28 19 NdNdNdNdNdNeNe NgNdNgNdNgNdNdNdNd 483294 69 5-9-2 Full deoxy gdgdg ee 30 19 NdNdNdNdNdNeNe NgNgNdNdNdNdNdNdNd 483438 81 2-10-4 Full deoxy gg eeee 23 19 NdNdNdNeNeNeNe NgNgNdNdNdNdNdNdNd 483444 84 2-10-4 Full deoxy gg eeee 28 19 NdNdNdNeNeNeNe NgNgNdNdNdNdNdNdNd 483448 77 2-10-4 Full deoxy gg eeee 30 19 NdNdNdNeNeNeNe NgNgNdNdNdNdNdNdNd 482847 79 2-10-2 Full deoxy gg gg 31 20 NdNdNdNgNg NgNgNdNdNdNdNdNdNd 482747 85 2-10-2 Full deoxy gg gg 32 20 NdNdNdNgNg NgNgNdNdNdNdNdNdNd 482873 81 2-10-2 Full deoxy gg gg 40 20 NdNdNdNgNg NgNgNdNdNdNdNdNdNdNd 482874 82 2-10-2 Full deoxy gg gg 75 20 NdNdNgNg NgNgNdNdNdNdNdNd 482875 82 2-10-2 Full deoxy gg gg 76 20 NdNdNdNdNgNg NgNgNgNdNdNdNdNd 482896 95 3-10-3 Full deoxy ggg ggg 77 19 NdNdNdNdNdNgNgNg NgNgNgNdNdNdNdNdNd 483019 89 3-10-3 Full deoxy ggg ggg 38 19 NdNdNdNdNgNgNg NgNdNgNdNdNdNdNdNd 483045 92 3-10-3 Full deoxy gdg gdg 77 19 NdNdNdNdNgNdNg NgNdNgNdNgNdNdNdNd 483194 64 3-10-3 Full deoxy gdg gdg 77 19 NdNdNdNdNdNeNe NgNdNgNdNgNdNdNdNd 483317 79 5-9-2 Full deoxy gdgdg ee 38 19 NdNdNdNdNdNeNe NgNgNdNdNdNdNdNdNd 483343 75 2-10-4 Full deoxy gg eeee 57 19 NdNdNdNeNeNeNe NgNgNdNdNdNdNdNdNdNdN 483471 76 2-10-4 Full deoxy gg eeee 38 19 dNdNeNeNeNe NgNgNdNdNdNdNdNdNd 483478 20 2-10-4 Full deoxy gg eeee 78 19 NdNdNdNeNeNeNe NeNeNeNeNeNdNdNdNdNd 407939 30 5-10-5 Full deoxy eeeee eeeee 72 21 NdNdNdNdNdNeNeNeNeNe NgNgNdNdNdNdNdNd 482784 83 2-10-2 Full deoxy gg gg 79 20 NdNdNdNdNgNg NgNgNdNdNdNdNdNd 482794 91 2-10-2 Full deoxy gg gg 54 20 NdNdNdNdNgNg NgNgNdNdNdNdNdNd 482804 80 2-10-2 Full deoxy gg gg 58 20 NdNdNdNdNgNg NgNgNdNdNdNdNdNd 482812 81 2-10-2 Full deoxy gg gg 66 20 NdNdNdNdNgNg NgNgNdNdNdNdNdNd 482813 92 2-10-2 Full deoxy gg gg 68 20 NdNdNdNdNgNg NgNgNdNdNdNdNdNd 482814 94 2-10-2 Full deoxy gg gg 70 20 NdNdNdNdNgNg NgNgNdNdNdNdNdNd 482815 81 2-10-2 Full deoxy gg gg 80 20 NdNdNdNdNgNg NgNgNdNdNdNdNdNd 482816 71 2-10-2 Full deoxy gg gg 74 20 NdNdNdNdNgNg NgNgNgNdNdNdNdNd 482916 90 3-10-3 Full deoxy ggg ggg 44 19 NdNdNdNdNdNgNgNg NgNgNgNdNdNdNdNd 482932 89 3-10-3 Full deoxy ggg ggg 48 19 NdNdNdNdNdNgNgNg NgNgNgNdNdNdNdNd 482953 93 3-10-3 Full deoxy ggg ggg 57 19 NdNdNdNdNdNgNgNg NgNgNgNdNdNdNdNd 482962 97 3-10-3 Full deoxy ggg ggg 67 19 NdNdNdNdNdNgNgNg NgNgNgNdNdNdNdNd 482963 96 3-10-3 Full deoxy ggg ggg 69 19 NdNdNdNdNdNgNgNg NgNgNgNdNdNdNdNd 482965 89 3-10-3 Full deoxy ggg ggg 73 19 NdNdNdNdNdNgNgNg NgNdNgNdNdNdNdNd 483065 69 3-10-3 Full deoxy ggg ggg 44 19 NdNdNdNdNdNgNdNg NgNdNgNdNdNdNdNd 483092 89 3-10-3 Full deoxy gdg gdg 53 19 NdNdNdNdNdNgNdNg NgNdNgNdNgNdNdNd 483241 79 5-9-2 Full deoxy gdgdg ee 53 19 NdNdNdNdNdNdNeNe NgNdNgNdNgNdNdNd 483253 76 5-9-2 Full deoxy gdgdg ee 59 19 NdNdNdNdNdNdNeNe NgNdNgNdNgNdNdNd 483258 70 5-9-2 Full deoxy gdgdg ee 64 19 NdNdNdNdNdNdNeNe NgNdNgNdNgNdNdNd 483260 62 5-9-2 Full deoxy gdgdg ee 67 19 NdNdNdNdNdNdNeNe NgNdNgNdNgNdNdNd 483261 76 5-9-2 Full deoxy gdgdg ee 69 19 NdNdNdNdNdNdNeNe NgNdNgNdNgNdNdNd 483262 75 5-9-2 Full deoxy gdgdg ee 71 19 NdNdNdNdNdNdNeNe NgNdNgNdNgNdNdNd 483263 73 5-9-2 Full deoxy gdgdg ee 73 19 NdNdNdNdNdNdNeNe NgNgNdNdNdNdNdNd 483364 78 2-10-4 Full deoxy gg eeee 81 19 NdNdNdNdNeNeNeNe NgNgNdNdNdNdNdNd 483395 86 2-10-4 Full deoxy gg eeee 53 19 NdNdNdNdNeNeNeNe NgNgNdNdNdNdNdNd 483413 83 2-10-4 Full deoxy gg eeee 65 19 NdNdNdNdNeNeNeNe NgNgNdNdNdNdNdNd 483414 76 2-10-4 Full deoxy gg eeee 67 19 NdNdNdNdNeNeNeNe NgNgNdNdNdNdNdNd 483415 85 2-10-4 Full deoxy gg eeee 69 19 NdNdNdNdNeNeNeNe NgNgNdNdNdNdNdNd 483416 77 2-10-4 Full deoxy gg eeee 71 19 NdNdNdNdNeNeNeNe NgNgNdNdNdNdNdNd 483417 83 2-10-4 Full deoxy gg eeee 73 19 NdNdNdNdNeNeNeNe e = 2′-MOE, d = 2′-deoxyribonucleoside, g = F-HNA

Example 3 Modified Antisense Oligonucleotides Comprising 2′-MOE and Constrained Ethyl (cEt) Modifications Targeting Human Target-X

Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. ISIS 403052, ISIS 407594, ISIS 407606, ISIS 407939, and ISIS 416438, which were described in an earlier publication (WO 2009/061851) were also tested.

The newly designed chimeric antisense oligonucleotides are 16 nucleotides in length and their motifs are described in Table 13. The chemistry column of Table 12 presents the sugar motif of each oligonucleotide, wherein “e” indicates a 2′-O-methythoxylethyl (2′-MOE) nucleoside, “k” indicates a constrained ethyl (cEt) and “d” indicates a 2′-deoxyribonucleoside. The internucleoside linkages throughout each gapmer are hosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.

Each gapmer listed in Table 13 is targeted to the human Target-X genomic sequence.

Activity of the newly designed gapmers was compared to ISIS 403052, ISIS 407594, ISIS 407606, ISIS 407939, and ISIS 416438. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Target-X, relative to untreated control cells. A total of 380 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 13. Each of the newly designed antisense oligonucleotides provided in Table 13 achieved greater than 64% inhibition and, therefore, are more potent than each of ISIS 403052, ISIS 407594, ISIS 407606, ISIS 407939, and ISIS 416438.

TABLE 13 Inhibition of human Target-X mRNA levels by chimeric antisense oligonucleotides targeted to Target-X ISIS % SEQ No Chemistry Motif inhibition CODE 403052 eeeee-(d10)-eeeee 5-10-5 64 82 407594 eeeee-(d10)-eeeee 5-10-5 40 83 407606 eeeee-(d10)-eeeee 5-10-5 39 84 407939 eeeee-(d10)-eeeee 5-10-5 57 72 416438 eeeee-(d10)-eeeee 5-10-5 62 85 484487 kdk-(d10)-dkdk 3-10-3 91 77 484539 kdk-d(10)-kdk 3-10-3 92 53 484546 kdk-d(10)-kdk 3-10-3 92 86 484547 kdk-d(10)-kdk 3-10-3 89 87 484549 kdk-d(10)-kdk 3-10-3 91 57 484557 kdk-d(10)-kdk 3-10-3 92 65 484558 kdk-d(10)-kdk 3-10-3 94 67 484559 kdk-d(10)-kdk 3-10-3 90 69 484582 kdk-d(10)-kdk 3-10-3 88 23 484632 kk-d(10)-eeee 2-10-4 90 88 484641 kk-d(10)-eeee 2-10-4 91 77 484679 kk-d(10)-eeee 2-10-4 90 49 484693 kk-d(10)-eeee 2-10-4 93 53 484711 kk-d(10)-eeee 2-10-4 92 65 484712 kk-d(10)-eeee 2-10-4 92 67 484713 kk-d(10)-eeee 2-10-4 85 69 484714 kk-d(10)-eeee 2-10-4 83 71 484715 kk-d(10)-eeee 2-10-4 93 73 484736 kk-d(10)-eeee 2-10-4 89 23 484742 kk-d(10)-eeee 2-10-4 93 28 484746 kk-d(10)-eeee 2-10-4 88 30 484771 kk-d(10)-eeee 2-10-4 89 89 e = 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside

Example 4 Antisense Inhibition of Human Target-X with 5-10-5 2′-MOE Gapmers

Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. Also tested were ISIS 403094, ISIS 407641, ISIS 407643, ISIS 407662, ISIS 407900, ISIS 407910, ISIS 407935, ISIS 407936, ISIS 407939, ISIS 416446, ISIS 416449, ISIS 416455, ISIS 416472, ISIS 416477, ISIS 416507, ISIS 416508, ISIS 422086, ISIS 422087, ISIS 422140, and ISIS 422142, 5-10-5 2′-MOE gapmers targeting human Target-X, which were described in an earlier publication (WO 2009/061851), incorporated herein by reference.

The newly designed modified antisense oligonucleotides are 20 nucleotides in length and their motifs are described in Tables 14 and 15. The chemistry column of Tables 14 and 15 present the sugar motif of each oligonucleotide, wherein “e” indicates a 2′-O-methythoxylethyl (2′-MOE) nucleoside and “d” indicates a 2′-deoxyribonucleoside. The internucleoside linkages throughout each gapmer are hosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.

Each gapmer listed in Table 14 is targeted to the human Target-X genomic sequence.

Activity of the newly designed gapmers was compared to ISIS 403094, ISIS 407641, ISIS 407643, ISIS 407662, ISIS 407900, ISIS 407910, ISIS 407935, ISIS 407936, ISIS 407939, ISIS 416446, ISIS 416449, ISIS 416455, ISIS 416472, ISIS 416477, ISIS 416507, ISIS 416508, ISIS 422086, ISIS 422087, ISIS 422140, and ISIS 422142. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Target-X, relative to untreated control cells. A total of 916 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Tables 14 and 15.

TABLE 14 Inhibition of human Target-X mRNA levels by chimeric antisense oligonucleotides targeted to Target-X ISIS No Chemistry % inhibition SEQ CODE 490275 e5-d(10)-e5 35 90 490277 e5-d(10)-e5 73 91 490278 e5-d(10)-e5 78 92 490279 e5-d(10)-e5 66 93 490323 e5-d(10)-e5 65 94 490368 e5-d(10)-e5 78 95 490396 e5-d(10)-e5 76 96 416507 e5-d(10)-e5 73 97 422140 e5-d(10)-e5 59 98 422142 e5-d(10)-e5 73 99 416508 e5-d(10)-e5 75 100 490424 e5-d(10)-e5 57 101 490803 e5-d(10)-e5 70 102 416446 e5-d(10)-e5 73 103 416449 e5-d(10)-e5 33 104 407900 e5-d(10)-e5 66 105 490103 e5-d(10)-e5 87 106 416455 e5-d(10)-e5 42 107 407910 e5-d(10)-e5 25 108 490149 e5-d(10)-e5 82 109 403094 e5-d(10)-e5 60 110 416472 e5-d(10)-e5 78 111 407641 e5-d(10)-e5 64 112 416477 e5-d(10)-e5 25 113 407643 e5-d(10)-e5 78 114 490196 e5-d(10)-e5 81 115 490197 e5-d(10)-e5 85 116 490208 e5-d(10)-e5 89 117 490209 e5-d(10)-e5 81 118 422086 e5-d(10)-e5 90 119 407935 e5-d(10)-e5 91 120 422087 e5-d(10)-e5 89 121 407936 e5-d(10)-e5 80 122 407939 e5-d(10)-e5 67 72 e = 2′-MOE, d = 2′-deoxynucleoside

TABLE 15 Inhibition of human Target-X mRNA levels by chimeric antisense oligonucleotides targeted to Target-X ISIS No Motif % inhibition SEQ CODE 407662 e5-d(10)-e5 76 123 416446 e5-d(10)-e5 73 103 e = 2′-MOE, d = 2′-deoxynucleoside

Example 5 Modified Chimeric Antisense Oligonucleotides Comprising Constrained Ethyl (cEt) Modifications at 5′ and 3′ Wing Regions Targeting human Target-X

Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. ISIS 407939, which was described in an earlier publication (WO 2009/061851) were also tested. ISIS 457851, ISIS 472925, ISIS 472926, ISIS 472935, ISIS 472942, ISIS 472958, ISIS 472959, ISIS 472970, ISIS 472976, ISIS 472983, ISIS 472984, ISIS 472988, ISIS 472991, ISIS 472994, ISIS 472995, ISIS 472996, ISIS 472998, and ISIS 473020, described in the Examples above were also included in the screen.

The newly designed chimeric antisense oligonucleotides in Table 16 were designed as 2-10-2 cEt gapmers. The newly designed gapmers are 14 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxyribonucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment comprises constrained ethyl (cEt) modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.

Each gapmer listed in Table 16 is targeted to the human Target-X genomic sequence.

Activity of the newly designed oligonucleotides was compared to ISIS 407939. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Target-X, relative to untreated control cells. A total of 614 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 16. Many of the newly designed antisense oligonucleotides provided in Table 16 achieved greater than 72% inhibition and, therefore, are more potent than ISIS 407939.

TABLE 16 Inhibition of human Target-X mRNA levels by chimeric antisense oligonucleotides targeted to Target-X % Wing SEQ ISIS No inhibition Motif Chemistry CODE 407939 72 5-10-5 cEt 72 473020 90 2-10-2 cEt 25 492465 83 2-10-2 cEt 124 492467 74 2-10-2 cEt 125 492492 84 2-10-2 cEt 126 492494 91 2-10-2 cEt 127 492503 89 2-10-2 cEt 128 492530 91 2-10-2 cEt 129 492534 91 2-10-2 cEt 130 492536 90 2-10-2 cEt 131 492541 84 2-10-2 cEt 132 492545 89 2-10-2 cEt 133 492566 90 2-10-2 cEt 134 492571 82 2-10-2 cEt 135 492572 89 2-10-2 cEt 136 492573 90 2-10-2 cEt 137 492574 92 2-10-2 cEt 138 492575 88 2-10-2 cEt 139 492593 83 2-10-2 cEt 140 492617 91 2-10-2 cEt 141 492618 92 2-10-2 cEt 142 492619 90 2-10-2 cEt 143 492621 75 2-10-2 cEt 144 492104 89 2-10-2 cEt 145 492105 86 2-10-2 cEt 146 492189 88 2-10-2 cEt 147 492194 92 2-10-2 cEt 148 492195 90 2-10-2 cEt 149 472925 87 2-10-2 cEt 32 492196 91 2-10-2 cEt 150 472926 88 2-10-2 cEt 33 492205 92 2-10-2 cEt 151 492215 77 2-10-2 cEt 152 492221 79 2-10-2 cEt 153 472935 82 2-10-2 cEt 36 492234 86 2-10-2 cEt 154 472942 85 2-10-2 cEt 43 492276 75 2-10-2 cEt 155 492277 75 2-10-2 cEt 156 492306 85 2-10-2 cEt 157 492317 93 2-10-2 cEt 158 472958 92 2-10-2 cEt 46 472959 88 2-10-2 cEt 47 492329 88 2-10-2 cEt 159 492331 95 2-10-2 cEt 160 492333 85 2-10-2 cEt 161 492334 88 2-10-2 cEt 162 457851 89 2-10-2 cEt 51 472970 92 2-10-2 cEt 52 492365 69 2-10-2 cEt 163 472976 94 2-10-2 cEt 54 472983 76 2-10-2 cEt 55 472984 72 2-10-2 cEt 56 492377 70 2-10-2 cEt 164 492380 80 2-10-2 cEt 165 492384 61 2-10-2 cEt 166 472988 59 2-10-2 cEt 60 492388 70 2-10-2 cEt 167 492389 70 2-10-2 cEt 168 492390 89 2-10-2 cEt 169 492391 80 2-10-2 cEt 170 472991 84 2-10-2 cEt 62 492398 88 2-10-2 cEt 171 492399 94 2-10-2 cEt 172 492401 91 2-10-2 cEt 173 492403 78 2-10-2 cEt 174 472994 95 2-10-2 cEt 66 472995 91 2-10-2 cEt 68 492404 84 2-10-2 cEt 175 492405 87 2-10-2 cEt 176 472996 85 2-10-2 cEt 70 492406 43 2-10-2 cEt 177 472998 92 2-10-2 cEt 74 492440 89 2-10-2 cEt 178

Example 6 Modified Chimeric Antisense Oligonucleotides Comprising Constrained Ethyl (cEt) Modifications at 5′ and 3′ Wing Regions Targeting Human Target-X

Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. Also tested was ISIS 407939, a 5-10-5 MOE gapmer targeting human Target-X, which was described in an earlier publication (WO 2009/061851). ISIS 472998 and ISIS 473046, described in the Examples above were also included in the screen.

The newly designed chimeric antisense oligonucleotides in Table 17 were designed as 2-10-2 cEt gapmers. The newly designed gapmers are 14 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxyribonucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment comprise constrained ethyl (cEt) modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.

Each gapmer listed in Table 17 is targeted to the human Target-X genomic sequence.

Activity of the newly designed gapmers was compared to ISIS 407939. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Target-X, relative to untreated control cells. A total of 757 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 17. Each of the newly designed antisense oligonucleotides provided in Table 17 achieved greater than 67% inhibition and, therefore, are more potent than 407939.

TABLE 17 Inhibition of human Target-X mRNA levels by chimeric antisense oligonucleotides targeted to Target-X ISIS % Wing SEQ No inhibition Motif chemistry CODE 407939 67 5-10-5 cEt 72 492651 77 2-10-2 cEt 179 492652 84 2-10-2 cEt 180 492658 87 2-10-2 cEt 181 492725 74 2-10-2 cEt 182 492730 78 2-10-2 cEt 183 492731 72 2-10-2 cEt 184 492784 72 2-10-2 cEt 185 492816 70 2-10-2 cEt 186 492818 73 2-10-2 cEt 187 492877 83 2-10-2 cEt 188 492878 79 2-10-2 cEt 189 492913 73 2-10-2 cEt 190 492914 82 2-10-2 cEt 191 492928 76 5-10-5 cEt 192 492938 80 2-10-2 cEt 193 492991 91 2-10-2 cEt 194 492992 73 2-10-2 cEt 195 493087 81 2-10-2 cEt 196 493114 80 2-10-2 cEt 197 493178 86 2-10-2 cEt 198 493179 69 2-10-2 cEt 199 493182 79 2-10-2 cEt 200 493195 71 2-10-2 cEt 201 473046 79 2-10-2 cEt 35 493201 86 2-10-2 cEt 202 493202 76 2-10-2 cEt 203 493255 80 2-10-2 cEt 204 493291 84 2-10-2 cEt 205 493292 90 2-10-2 cEt 206 493296 82 2-10-2 cEt 207 493298 77 2-10-2 cEt 208 493299 76 5-10-5 cEt 209 493304 77 2-10-2 cEt 210 493312 75 2-10-2 cEt 211 493333 76 2-10-2 cEt 212 472998 85 2-10-2 cEt 74

Example 7 Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B Cells

Antisense oligonucleotides from the studies above, exhibiting in vitro inhibition of Target-X mRNA, were selected and tested at various doses in Hep3B cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.67 μM, 2.00 μM, 1.11 μM, and 6.00 μM concentrations of antisense oligonucleotide, as specified in Table 18. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human Target-X primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented in Table 18. As illustrated in Table 18, Target-X mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. The data also confirms that several of the newly designed gapmers are more potent than ISIS 407939 of the previous publication.

TABLE 18 Dose-dependent antisense inhibition of human Target-X in Hep3B cells using electroporation 666.6667 2000.0 6000.0 IC₅₀ ISIS No nM nM nM (μM) 407939 47 68 85 0.7 457851 60 80 93 <0.6 472916 53 80 87 <0.6 472925 62 86 95 <0.6 472926 66 77 85 <0.6 472935 54 84 94 <0.6 472958 66 82 88 <0.6 472959 64 81 93 <0.6 472970 72 87 86 <0.6 472976 78 92 97 <0.6 472994 79 92 96 <0.6 472995 61 82 93 <0.6 472996 73 91 95 <0.6 472998 63 90 95 <0.6 473019 55 80 86 <0.6 473020 61 76 85 <0.6 473046 61 80 94 <0.6 473055 55 84 94 <0.6 492104 53 76 88 <0.6 492105 62 80 90 <0.6 492189 57 80 92 <0.6 492194 57 83 91 <0.6 492195 58 81 95 <0.6 492196 62 86 95 <0.6 492205 62 87 95 <0.6 492215 60 78 89 <0.6 492221 63 76 92 <0.6 492234 51 74 91 0.5 492276 50 56 95 0.8 492277 58 73 81 <0.6 492306 61 75 84 <0.6 492317 59 80 93 <0.6 492329 59 70 89 <0.6 492331 69 87 95 <0.6 492333 47 70 85 0.7 492334 57 77 90 <0.6 492390 72 88 95 <0.6 492399 68 91 96 <0.6 492401 68 89 95 <0.6 492404 65 87 94 <0.6 492405 44 81 90 0.7 492406 65 82 92 <0.6 492440 50 70 89 0.6 492465 16 80 79 1.4 492467 58 77 92 <0.6 492492 45 80 94 0.7 492494 63 82 93 <0.6 492503 55 81 93 <0.6 492530 70 86 90 <0.6 492534 67 85 91 <0.6 492536 54 81 89 <0.6 492541 54 71 85 <0.6 492545 59 78 89 <0.6 492566 59 84 85 <0.6 492571 52 81 89 <0.6 492572 67 83 90 <0.6 492573 69 83 92 <0.6 492574 65 82 91 <0.6 492575 72 83 91 <0.6 492593 61 78 90 <0.6 492617 62 80 93 <0.6 492618 47 79 94 0.6 492619 54 82 95 <0.6 492621 44 85 92 0.6 492651 53 66 91 0.6 492652 61 78 88 <0.6 492658 59 79 88 <0.6 492725 43 84 89 0.6 192730 51 87 93 0.1 492731 46 82 90 0.6 492784 56 88 96 <0.6 492816 68 89 97 <0.6 492818 64 84 96 <0.6 492877 67 91 93 <0.6 492878 80 89 93 <0.6 492913 53 87 92 <0.6 492914 75 89 96 <0.6 492928 60 83 94 <0.6 492938 70 90 92 <0.6 492991 67 93 99 <0.6 492992 0 82 95 2.1 493087 54 81 90 <0.6 493114 50 73 90 0.6 493178 71 88 96 <0.6 493179 47 82 95 0.6 493182 79 87 91 <0.6 493195 55 78 90 <0.6 493201 87 93 96 <0.6 493202 68 89 94 <0.6 493255 57 79 93 <0.6 493291 57 87 93 <0.6 493292 70 89 93 <0.6 493296 35 84 91 0.9 493298 57 84 92 <0.6 493299 65 84 93 <0.6 493304 68 86 94 <0.6 493312 53 82 91 <0.6 493333 66 84 87 <0.6

Example 8 Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B Cells

Additional antisense oligonucleotides from the studies described above, exhibiting in vitro inhibition of Target-X mRNA, were selected and tested at various doses in Hep3B cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.67 μM, 2.00 μM, 1.11 μM, and 6.00 μM concentrations of antisense oligonucleotide, as specified in Table 19. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human Target-X primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells. As illustrated in Table 19, Target-X mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. The data also confirms that several of the newly designed gapmers are more potent than ISIS 407939.

TABLE 19 Dose-dependent antisense inhibition of human Target-X in Hep3B cells using electroporation 0.67 2.00 6.00 IC₅₀ ISIS No μM μM μM (μM) 407939 52 71 86 0.6 472983 49 83 97 0.5 472984 51 82 95 0.5 472991 49 82 95 0.5 472998 59 88 96 <0.6 492365 74 91 96 <0.6 492377 56 76 91 <0.6 492380 63 79 95 <0.6 492384 67 84 94 <0.6 492388 69 87 97 <0.6 492389 62 90 96 <0.6 492391 56 84 94 <0.6 492398 63 80 95 <0.6 492403 58 81 91 <0.6

Example 9 Modified Chimeric Antisense Oligonucleotides Comprising 2′-methoxyethyl (2′-MOE) Modifications at 5′ and 3′ Wing Regions Targeting Human Target-X

Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. Also tested were ISIS 403052, ISIS 407939, ISIS 416446, ISIS 416472, ISIS 416507, ISIS 416508, ISIS 422087, ISIS 422096, ISIS 422130, and ISIS 422142 which were described in an earlier publication (WO 2009/061851), incorporated herein by reference. ISIS 490149, ISIS 490197, ISIS 490209, ISIS 490275, ISIS 490277, and ISIS 490424, described in the Examples above, were also included in the screen.

The newly designed chimeric antisense oligonucleotides in Table 20 were designed as 3-10-4 2′-MOE gapmers. These gapmers are 17 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxyribonucleosides and is flanked by wing segments on the 5′ direction with three nucleosides and the 3′ direction with four nucleosides. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has 2′-MOE modifications. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.

Each gapmer listed in Table 20 is targeted to the human Target-X genomic sequence.

Activity of the newly designed oligonucleotides was compared to ISIS 403052, ISIS 407939, ISIS 416446, ISIS 416472, ISIS 416507, ISIS 416508, ISIS 422087, ISIS 422096, ISIS 422130, and ISIS 422142. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells. A total of 272 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 20. Several of the newly designed antisense oligonucleotides provided in Table 19 are more potent than antisense oligonucleotides from the previous publication.

TABLE 20 Inhibition of human Target-X mRNA levels by chimeric antisense oligonucleotides targeted to Target-X ISIS % Wing SEQ No inhibition Motif Chemistry CODE 403052 51 5-10-5 2′-MOE 82 407939 78 5-10-5 2′-MOE 72 416446 70 5-10-5 2′-MOE 103 416472 79 5-10-5 2′-MOE 111 416507 84 5-10-5 2′-MOE 97 416508 80 5-10-5 2′-MOE 100 422087 89 5-10-5 2′-MOE 121 422096 78 5-10-5 2′-MOE 219 422130 81 5-10-5 2′-MOE 225 422142 84 5-10-5 2′-MOE 99 490275 77 5-10-5 2′-MOE 90 513462 79 3-10-4 2′-MOE 213 513463 81 3-10-4 2′-MOE 214 490277 74 5-10-5 2′-MOE 91 513487 83 3-10-4 2′-MOE 215 513504 81 3-10-4 2′-MOE 216 513507 86 3-10-4 2′-MOE 217 513508 85 3-10-4 2′-MOE 218 490424 69 5-10-5 2′-MOE 101 491122 87 5-10-5 2′-MOE 220 513642 79 3-10-4 2′-MOE 221 490149 71 5-10-5 2′-MOE 109 513419 90 3-10-4 2′-MOE 222 513420 89 3-10-4 2′-MOE 223 513421 88 3-10-4 2′-MOE 224 490197 77 5-10-5 2′-MOE 116 513446 89 3-10-4 2′-MOE 226 513447 83 3-10-4 2′-MOE 227 490209 79 5-10-5 2′-MOE 118 513454 84 3-10-4 2′-MOE 228 513455 92 3-10-4 2′-MOE 229 513456 89 3-10-4 2′-MOE 230 513457 83 3-10-4 2′-MOE 231

Example 10 Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B Cells

Antisense oligonucleotides from the studies above, exhibiting in vitro inhibition of Target-X mRNA, were selected and tested at various doses in Hep3B cells. ISIS 403052, ISIS 407643, ISIS 407935, ISIS 407936, ISIS 407939, ISIS 416446, ISIS 416459, ISIS 416472, ISIS 416507, ISIS 416508, ISIS 416549, ISIS 422086, ISIS 422087, ISIS 422130, ISIS and 422142, 5-10-5 MOE gapmers targeting human Target-X, which were described in an earlier publication (WO 2009/061851).

Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.625 μM, 1.25 μM, 2.50 μM, 5.00 μM and 10.00 μM concentrations of antisense oligonucleotide, as specified in Table 21. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human Target-X primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented in Table 21. As illustrated in Table 21, Target-X mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. The data also confirms that the newly designed gapmers are potent than gapmers from the previous publication.

TABLE 21 Dose-dependent antisense inhibition of human Target-X in Hep3B cells using electroporation ISIS 0.625 1.25 2.50 5.00 10.00 IC₅₀ No μM μM μM μM μM (μM) 403052 21 35 63 82 89 1.9 407643 29 46 67 83 90 1.4 407935 52 68 80 89 91 <0.6 407936 31 51 62 78 84 1.4 407939 30 61 74 83 88 1.0 416446 37 53 64 76 83 1.2 416459 51 76 83 90 92 <0.6 416472 37 52 66 78 85 1.2 416507 45 68 82 87 90 0.7 416508 33 56 74 84 89 1.1 416549 57 71 78 82 85 <0.6 422086 46 67 77 89 92 0.7 422087 50 69 74 86 91 0.6 422130 32 65 78 92 93 0.9 422142 59 73 84 86 88 <0.6 490103 52 57 66 83 88 0.9 490149 34 58 71 85 91 1.0 490196 26 59 66 79 84 1.3 490197 39 63 74 81 90 0.8 490208 44 70 76 83 88 0.6 490275 36 58 76 85 89 1.0 490277 37 63 73 87 87 0.8 490279 40 54 72 83 89 1.0 490323 49 68 79 86 90 <0.6 490368 39 62 76 86 91 0.8 490396 36 53 69 80 87 1.1 490424 45 65 69 76 82 0.6 490803 57 74 85 89 92 <0.6 513419 60 71 85 95 96 <0.6 513420 37 69 79 94 96 0.7 513421 46 64 84 95 97 0.6 513446 47 81 88 95 96 <0.6 513447 56 74 81 92 96 <0.6 513454 50 77 82 93 95 <0.6 513455 74 82 91 96 96 <0.6 513456 66 80 88 94 95 <0.6 513457 54 67 80 87 89 <0.6 513462 49 72 84 87 89 <0.6 513463 36 62 76 85 89 0.9 513487 42 56 73 87 93 0.9 513504 47 65 81 90 91 0.6 513505 39 50 78 85 92 1.0 513507 52 73 83 89 93 <0.6 513508 56 78 85 91 94 <0.6

Example 11 Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B Cells

Additional antisense oligonucleotides from the studies above, exhibiting in vitro inhibition of Target-X mRNA, were tested at various doses in Hep3B cells. ISIS 407935, ISIS 407939, ISIS 416446, ISIS 416472, ISIS 416507, ISIS 416549, ISIS 422086, ISIS 422087, ISIS 422096, and ISIS 422142 5-10-5 MOE gapmers targeting human Target-X, which were described in an earlier publication (WO 2009/061851).

Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.3125 μM, 0.625 μM, 1.25 μM, 2.50 μM, 5.00 μM and 10.00 μM concentrations of antisense oligonucleotide, as specified in Table 22. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human Target-X primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells. As illustrated in Table 22, Target-X mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. The data also confirms that the newly designed gapmers are more potent than gapmers from the previous publication.

TABLE 22 Dose-dependent antisense inhibition of human Target-X in Hep3B cells using electroporation ISIS 0.3125 0.625 1.250 2.500 5.000 10.000 IC₅₀ No μM μM μM μM μM μM (μM) 407935 30 49 75 86 91 94 0.6 407939 30 48 61 78 85 90 0.8 416446 27 52 63 75 85 90 0.7 416472 38 51 72 83 88 94 0.5 416507 58 81 76 84 89 92 <0.3 416549 52 67 75 81 88 89 0.3 422086 48 49 68 78 86 91 0.5 422087 30 56 66 83 72 92 0.6 422096 47 63 70 77 83 85 <0.3 422142 69 85 87 85 89 91 <0.3 490103 52 57 68 78 87 93 0.4 490149 33 64 62 77 86 93 0.5 490197 38 46 60 75 87 93 0.7 490208 46 62 73 83 88 91 0.4 490209 40 54 72 79 85 94 0.5 490275 57 61 67 78 85 91 0.3 490277 33 59 77 79 91 94 0.5 490323 43 61 72 69 84 87 0.4 490368 50 64 78 83 90 92 <0.3 490396 46 64 68 84 84 90 0.3 490424 24 47 58 72 76 82 1.0 490803 45 60 70 84 88 89 0.3 513419 32 53 76 88 93 95 0.5 513420 35 59 72 82 94 97 0.5 513421 46 67 78 86 94 96 <0.3 513116 26 61 77 89 91 97 0.5 513447 22 48 60 82 91 95 0.8 513454 25 59 76 86 94 96 0.5 513455 60 73 85 89 95 96 <0.3 513456 49 60 81 88 94 95 <0.3 513457 43 50 72 77 87 92 0.5 513462 25 48 58 76 83 88 0.8 513463 22 45 66 73 85 88 0.9 513487 41 56 65 79 86 90 0.4 513504 19 48 63 76 87 92 0.9 513505 11 21 54 73 85 90 1.4 513507 47 55 72 82 90 91 0.3 513508 31 59 74 85 92 93 0.5 513642 43 55 67 80 88 92 0.4

Example 12 Tolerability of 2′-MOE Gapmers Targeting Human Target-X in BALB/c Mice

BALB/c mice are a multipurpose mice model, frequently utilized for safety and efficacy testing. The mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

Groups of male BALB/c mice were injected subcutaneously twice a week for 3 weeks with 50 mg/kg of ISIS 407935, ISIS 416472, ISIS 416549, ISIS 422086, ISIS 422087, ISIS 422096, ISIS 422142, ISIS 490103, ISIS 490149, ISIS 490196, ISIS 490208, ISIS 490209, ISIS 513419, ISIS 513420, ISIS 513421, ISIS 513454, ISIS 513455, ISIS 513456, ISIS 513457, ISIS 513462, ISIS 513463, ISIS 513487, ISIS 513504, ISIS 513508, and ISIS 513642. One group of male BALB/c mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).

ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 407935, ISIS 416472, ISIS 416549, ISIS 422087, ISIS 422096, ISIS 490103, ISIS 490196, ISIS 490208, ISIS 513454, ISIS 513455, ISIS 513456, ISIS 513457, ISIS 513487, ISIS 513504, and ISIS 513508 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 422086, ISIS 490209, ISIS 513419, ISIS 513420, and ISIS 513463 were considered tolerable in terms of liver function.

Example 13 Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B Cells

Additional antisense oligonucleotides from the studies above, exhibiting in vitro inhibition of Target-X mRNA were selected and tested at various doses in Hep3B cells. Also tested was ISIS 407939, a 5-10-5 MOE gapmer, which was described in an earlier publication (WO 2009/061851).

Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.074 μM, 0.222 μM, 0.667 μM, 2.000 μM, and 6.000 μM concentrations of antisense oligonucleotide, as specified in Table 23. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human Target-X primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented in Table 23. As illustrated in Table 23, Target-X mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. Many of the newly designed antisense oligonucleotides provided in Table 23 achieved an IC₅₀ of less than 0.9 μM and, therefore, are more potent than ISIS 407939.

TABLE 23 Dose-dependent antisense inhibition of human Target-X in Hep3B cells using electroporation ISIS 0.074 0.222 0.667 2.000 6.000 IC₅₀ No μM μM μM μM μM (μM) 407939 2 17 53 70 87 0.9 472970 17 47 75 92 95 0.3 472988 0 8 21 54 92 1.4 472996 18 59 74 93 95 0.2 473244 91 95 97 99 99 <0.07 473286 6 53 85 92 98 0.3 473359 2 3 20 47 67 2.6 473392 71 85 88 92 96 <0.07 473393 91 96 97 98 99 <0.07 473547 85 88 93 97 98 <0.07 473567 0 25 66 88 95 0.7 473589 8 47 79 94 99 0.3 482814 23 68 86 93 96 0.1 482815 6 48 65 90 96 0.4 482963 3 68 85 94 96 0.2 483241 14 33 44 76 93 0.6 483261 14 21 41 72 88 0.7 483290 0 1 41 69 92 1.0 483414 8 1 36 76 91 0.9 483415 0 40 52 84 94 0.6 484559 26 51 78 87 97 0.2 484713 6 5 53 64 88 0.9

Example 14 Modified Antisense Oligonucleotides Comprising 2′-O-methoxyethyl (2′-MOE) and Constrained Ethyl (cEt) Modifications Targeting Human Target-X

Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. Also tested was ISIS 407939, a 5-10-5 MOE gapmer targeting human Target-X, which was described in an earlier publication (WO 2009/061851). ISIS 472998, ISIS 492878, and ISIS 493201 and 493182, 2-10-2 cEt gapmers, described in the Examples above were also included in the screen.

The newly designed modified antisense oligonucleotides are 16 nucleotides in length and their motifs are described in Table 24. The chemistry column of Table 24 presents the sugar motif of each oligonucleotide, wherein “e” indicates a 2′-O-methythoxylethyl (2′-MOE) nucleoside, “k” indicates a constrained ethyl (cEt) nucleoside and “d” indicates a 2′-deoxyribonucleoside. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.

Each gapmer listed in Table 24 is targeted to the human Target-X genomic sequence.

Activity of newly designed gapmers was compared to ISIS 407939. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells and demonstrate that several of the newly designed gapmers are more potent than ISIS 407939. A total of 685 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 24.

TABLE 24 Inhibition of human Target-X mRNA levels by chimeric antisense oligonucleotides targeted to Target-X % SEQ ISIS No inhibition Chemistry CODE 407939 68 eeeee-d(10)-eeeee 72 492878 73 kk-d(10)-kk 493182 80 kk-d(10)-kk 493201 84 kk-d(10)-kk 472998 91 kk-d(10)-kk 515640 75 eee-d(10)-kkk 23 515637 77 eee-d(10)-kkk 232 515554 72 eee-d(10)-kkk 233 515406 80 kkk-d(10)-eee 234 515558 81 eee-d(10)-kkk 234 515407 88 kkk-d(10)-eee 235 515408 85 kkk-d(10)-eee 236 515422 86 kkk-d(10)-eee 237 515423 90 kkk-d(10)-eee 238 515575 84 eee-d(10)-kkk 238 515424 87 kkk-d(10)-eee 239 515432 78 kkk-d(10)-eee 240 515433 71 kkk-d(10)-eee 241 515434 76 kkk-d(10)-eee 242 515334 85 kkk-d(10)-eee 243 515649 61 eee-d(10)-kkk 88 515338 86 kkk-d(10)-eee 244 515438 76 kkk-d(10)-eee 245 515439 75 kkk-d(10)-eee 246 516003 87 eee-d(10)-kkk 247 515647 60 eee-d(10)-kkk 77 515639 78 eee-d(10)-kkk 34 493201 84 eee-d(10)-kkk 202 515648 36 kkk-d(10)-eee 248 515641 69 kk-d(10)-eeee 39 515650 76 kkk-d(10)-eee 44 515354 87 eee-d(10)-kkk 249 515926 87 eee-d(10)-kkk 250 515366 87 kk-d(10)-eeee 251 515642 58 kkk-d(10)-eee 252 515643 81 eee-d(10)-kkk 53 515944 84 kk-d(10)-eeee 253 515380 90 kkk-d(10)-eee 254 515532 83 kkk-d(10)-eee 254 515945 85 kk-d(10)-eeee 254 515381 82 kk-d(10)-eeee 255 515382 95 kkk-d(10)-eee 256 515948 94 eee-d(10)-kkk 256 515949 87 eee-d(10)-kkk 257 515384 89 kkk-d(10)-eee 258 515635 82 kk-d(10)-eeee 65 515638 90 kkk-d(10)-eee 67 515386 92 kk-d(10)-eeee 259 515951 84 eee-d(10)-kkk 259 515387 78 kkk-d(10)-eee 260 515952 89 kkk-d(10)-eee 260 515636 90 kkk-d(10)-eee 69 515388 84 eee-d(10)-kkk 261 e = 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside

Example 15 Tolerability of Modified Oligonucleotides Comprising 2′-O-methoxyethyl (2′-MOE) and Constrained Ethyl (cEt) Modifications Targeting Human Target-X in BALB/c Mice

BALB/c mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.

Additionally, the newly designed modified antisense oligonucleotides were also added to this screen. The newly designed chimeric antisense oligonucleotides are 16 nucleotides in length and their motifs are described in Table 25. The chemistry column of Table 25 presents the sugar motif of each oligonucleotide, wherein “e” indicates a 2′-O-methythoxylethyl (2′-MOE) nucleoside, “k” indicates a constrained ethyl (cEt) nucleoside and “d” indicates a 2′-deoxynucleoside. The internucleoside linkages throughout each gapmer are hosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.

Each gapmer listed in Table 25 is targeted to either the human Target-X genomic sequence.

TABLE 25 Modified chimeric antisense oligonucleotides targeted to Target-X ISIS No Chemistry SEQ CODE 516044 eee-d(10)-kkk 21 516045 eee-d(10)-kkk 22 516058 eee-d(10)-kkk 26 516059 eee-d(10)-kkk 27 516060 eee-d(10)-kkk 28 516061 eee-d(10)-kkk 29 516062 eee-d(10)-kkk 30 516046 eee-d(10)-kkk 37 516063 eee-d(10)-kkk 38 516064 eee-d(10)-kkk 89 516065 eee-d(10)-kkk 262 516066 eee-d(10)-kkk 263 516047 eee-d(10)-kkk 41 516048 eee-d(10)-kkk 42 516049 eee-d(10)-kkk 81 516050 eee-d(10)-kkk 45 516051 eee-d(10)-kkk 48 516052 eee-d(10)-kkk 49 515652 eee-d(10)-kkk 50 508039 eee-d(10)-kkk 264 516053 eee-d(10)-kkk 265 515654 eee-d(10)-kkk 76 515656 eee-d(10)-kkk 77 516054 eee-d(10)-kkk 57 516055 eee-d(10)-kkk 59 515655 eee-d(10)-kkk 61 516056 eee-d(10)-kkk 63 516057 eee-d(10)-kkk 64 515653 eee-d(10)-kkk 71 515657 eee-d(10)-kkk 73 e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

Treatment

Groups of 4-6-week old male BALB/c mice were injected subcutaneously twice a week for 3 weeks with 50 mg/kg/week of ISIS 457851, ISIS 515635, ISIS 515636, ISIS 515637, ISIS 515638, ISIS 515639, ISIS 515640, ISIS 515641, ISIS 515642, ISIS 515643, ISIS 515647, ISIS 515648, ISIS 515649, ISSI 515650, ISIS 515652, ISIS 515653, ISIS 515654, ISIS 515655, ISIS 515656, ISIS 515657, ISIS 516044, ISIS 516045, ISIS 516046, ISIS 516047, ISIS 516048, ISIS 516049, ISIS 516050, ISIS 516051, ISIS 516052, ISIS 516053, ISIS 516054, ISIS 516055, ISIS 516056, ISIS 516057, ISIS 516058, ISIS 516059, ISIS 516060, ISIS 516061, ISIS 516062, ISIS 516063, ISIS 516064, ISIS 516065, and ISIS 516066. One group of 4-6-week old male BALB/c mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).

ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 515636, ISIS 515639, ISIS 515641, ISIS 515642, ISIS 515648, ISIS 515650, ISIS 515652, ISIS 515653, ISIS 515655, ISIS 515657, ISIS 516044, ISIS 516045, ISIS 516047, ISIS 516048, ISIS 516051, ISIS 516052, ISIS 516053, ISIS 516055, ISIS 516056, ISIS 516058, ISIS 516059, ISIS 516060, ISIS 516061, ISIS 516062, ISIS 516063, ISIS 516064, ISIS 516065, and ISIS 516066 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 457851, ISIS 515635, ISIS 515637, ISIS 515638, ISIS 515643, ISIS 515647, ISIS 515649, ISIS 515650, ISIS 515652, ISIS 515654, ISIS 515656, ISIS 516056, and ISIS 516057 were considered tolerable in terms of liver function.

Example 16 Efficacy of Modified Oligonucleotides Comprising 2′-O-methoxyethyl (2′-MOE) and Constrained Ethyl (cEt) Modifications Targeting Human Target-X in Transgenic Mice

Transgenic mice were developed at Taconic farms harboring a Target-X genomic DNA fragment. The mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for efficacy.

Treatment

Groups of 3-4 male and female transgenic mice were injected subcutaneously twice a week for 3 weeks with 20 mg/kg/week of ISIS 457851, ISIS 515636, ISIS 515639, ISIS 515653, ISIS 516053, ISIS 516065, and ISIS 516066. One group of mice was injected subcutaneously twice a week for 3 weeks with control oligonucleotide, ISIS 141923 (CCTTCCCTGAAGGTTCCTCC, 5-10-5 MOE gapmer with no known murine target, SEQ ID NO: 22). One group of mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

RNA Analysis

RNA was extracted from plasma for real-time PCR analysis of Target-X, using primer probe set RTS2927. The mRNA levels were normalized using RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to control. As shown in Table 26, each of the antisense oligonucleotides achieved reduction of human Target-X mRNA expression over the PBS control. Treatment with the control oligonucleotide did not achieve reduction in Target-X levels, as expected.

TABLE 26 Percent inhibition of Target-X mRNA in transgenic mice ISIS No % inhibition 141923 0 457851 76 515636 66 515639 49 515653 78 516053 72 516065 59 516066 39

Protein Analysis

Plasma protein levels of Target-X were estimated using a Target-X ELISA kit (purchased from Hyphen Bio-Med). Results are presented as percent inhibition of Target-X, relative to control. As shown in Table 27, several antisense oligonucleotides achieved reduction of human Target-X protein expression over the PBS control. ‘n.d.’ indicates that the value for that particular oligonucleotide was not measured.

TABLE 27 Percent inhibition of Target-X protein levels in transgenic mice ISIS No % inhibition 141923 0 457851 64 515636 68 515639 46 515653 0 516053 19 516065 0 516066 7

Example 17 Efficacy of Modified Oligonucleotides Comprising 2′-O-methoxyethyl (2′-MOE) and Constrained Ethyl (cEt) Modifications Targeting Human Target-X in Transgenic Mice

Transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for efficacy.

Treatment

Groups of 2-4 male and female transgenic mice were injected subcutaneously twice a week for 3 weeks with 10 mg/kg/week of ISIS 407935, ISIS 416472, ISIS 416549, ISIS 422087, ISIS 422096, ISIS 473137, ISIS 473244, ISIS 473326, ISIS 473327, ISIS 473359, ISIS 473392, ISIS 473393, ISIS 473547, ISIS 473567, ISIS 473589, ISIS 473630, ISIS 484559, ISIS 484713, ISIS 490103, ISIS 490196, ISIS 490208, ISIS 513419, ISIS 513454, ISIS 513455, ISIS 513456, ISIS 513457, ISIS 513487, ISIS 513508, ISIS 515640, ISIS 515641, ISIS 515642, ISIS 515648, ISIS 515655, ISIS 515657, ISIS 516045, ISIS 516046, ISIS 516047, ISIS 516048, ISIS 516051, ISIS 516052, ISIS 516055, ISIS 516056, ISIS 516059, ISIS 516061, ISIS 516062, and ISIS 516063. One group of mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Protein Analysis

Plasma protein levels of Target-X were estimated using a Target-X ELISA kit (purchased from Hyphen Bio-Med). Results are presented as percent inhibition of Target-X, relative to control. As shown in Table 28, several antisense oligonucleotides achieved reduction of human Target-X over the PBS control.

TABLE 28 Percent inhibition of Target-X plasma protein levels in transgenic mice ISIS No % inhibition 407935 80 416472 49 416549 29 422087 12 422096 21 473137 57 473244 67 473326 42 473327 100 473359 0 473392 22 473393 32 473547 73 473567 77 473589 96 473630 75 484559 75 484713 56 490103 0 490196 74 490208 90 513419 90 513454 83 513455 91 513456 81 513457 12 513487 74 513508 77 515640 83 515641 87 515642 23 515648 32 515655 79 515657 81 516045 52 516046 79 516047 65 516048 79 516051 84 516052 72 516055 70 516056 0 516059 39 516061 64 516062 96 516063 24

Example 18 Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B Cells

Antisense oligonucleotides exhibiting in vitro inhibition of Target-X mRNA were selected and tested at various doses in Hep3B cells. Also tested was ISIS 407939, a 5-10-5 MOE gapmer targeting human Target-X, which was described in an earlier publication (WO 2009/061851).

Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.074 μM, 0.222 μM, 0.667 μM, 2.000 μM, and 6.000 μM concentrations of antisense oligonucleotide, as specified in Table 29. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human Target-X primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented in Table 29. As illustrated in Table 29, Target-X mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. Many of the newly designed antisense oligonucleotides provided in Table 29 achieved an IC₅₀ of less than 2.0 μM and, therefore, are more potent than ISIS 407939.

TABLE 29 Dose-dependent antisense inlibition of human Target-X in Hep3B cells using electroporation ISIS 0.074 0.222 0.667 2.000 6.000 IC₅₀ No μM μM μM μM μM (μM) 407939 0 9 21 58 76 2.0 515636 14 32 50 62 81 0.7 515639 10 24 41 61 67 1.3 515640 4 16 35 52 63 2.0 515641 0 21 27 55 66 1.9 515642 3 13 36 44 66 2.2 515648 8 10 10 5 16 >6.0 515653 9 35 26 55 71 1.5 515655 0 0 6 13 42 >6.0 515657 0 13 17 38 51 6.0 516045 0 6 15 19 40 >6.0 516046 0 7 32 48 69 2.1 516047 12 27 41 50 63 1.8 516051 9 8 34 52 66 2.0 516052 17 42 27 53 75 1.2 516053 9 7 28 63 77 1.3 516055 0 3 27 54 75 2.0 516056 0 4 14 52 66 2.6 516057 0 34 33 51 70 1.6 516058 13 12 25 47 74 2.0 516059 4 15 36 47 68 1.9 516060 0 1 39 29 63 3.2 516061 0 0 24 0 3 <6.0 516062 0 20 43 65 78 1.0 516063 0 8 10 37 61 3.8 516064 0 3 13 45 69 2.7 516065 0 14 38 63 76 1.3 516066 0 3 30 55 75 1.7

Example 19 Modified Oligonucleotides Comprising 2′-O-methoxyethyl (2′-MOE) and Constrained Ethyl (cEt) Modifications Targeting Human Target-X

Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. ISIS 472998, ISIS 515652, ISIS 515653, ISIS 515654, ISIS 515655, ISIS 515656, and ISIS 515657, described in the Examples above were also included in the screen.

The newly designed chimeric antisense oligonucleotides are 16 or 17 nucleotides in length and their motifs are described in Table 30. The chemistry column of Table 30 presents the sugar motif of each oligonucleotide, wherein “e” indicates a 2′-O-methythoxylethyl (2′-MOE) nucleoside, “k” indicates a constrained ethyl (cEt) nucleoside and “d” indicates a 2′-deoxynucleoside. The internucleoside linkages throughout each gapmer are hosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.

Each gapmer listed in Table 30 is targeted to the human Target-X genomic sequence.

Activity of newly designed gapmers was compared to ISIS 407939. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells.

TABLE 30 Inhibition of human Target-X mRNA levels by chimeric antisense oligonucleotides targeted to Target-X ISIS % SEQ No inhibition Chemistry CODE 472998 85 kk-d(10)-kk 74 515652 63 eee-d(10)-kkk 50 515653 67 eee-d(10)-kkk 71 515654 78 eee-d(10)-kkk 86 515655 41 eee-d(10)-kkk 61 515656 74 eee-d(10)-kkk 87 515657 49 eee-d(10)-kkk 73 529265 52 eek-d(10)-keke 267 529332 82 eek-d(10)-keke 268 529334 78 eek-d(10)-keke 269 529186 85 eek-d(10)-keke 213 529223 81 eek-d(10)-kkke 213 529129 75 eee-d(10)-kkk 270 529149 82 kkk-d(10)-eee 270 529177 77 eek-d(10)-keke 214 529214 78 eek-d(10)-kkke 214 529178 79 eek-d(10)-keke 271 529215 82 eek-d(10)-kkke 271 529179 71 eek-d(10)-keke 272 529216 77 eek-d(10)-kkke 272 529193 69 eek-d(10)-keke 273 529230 70 eek-d(10)-kkke 273 529136 48 eee-d(10)-kkk 274 529156 68 kkk-d(10)-eee 274 529194 44 eek-d(10)-keke 275 529231 56 eek-d(10)-kkke 275 529137 34 eee-d(10)-kkk 276 529157 79 kkk-d(10)-eee 276 529336 57 eek-d(10)-keke 277 529338 73 eek-d(10)-keke 278 529195 55 eek-d(10)-keke 279 529232 68 eek-d(10)-kkke 279 529340 65 eek-d(10)-keke 280 529342 69 eek-d(10)-keke 281 529812 69 k-d(10)-kekee 282 529831 62 k-d(10)-kdkee 282 529733 64 ke-d(10)-keke 283 529753 52 ek-d(10)-keke 283 529773 57 ke-d(10)-kdke 283 529793 36 ek-d(10)-kdke 283 529862 48 kde-d(10)-kdke 284 529882 35 edk-d(10)-kdke 284 529902 44 k-(d4)-k-(d4)-k-(d4)-ke 284 529559 71 eek-d(10)-kke 26 529584 57 kee-d(10)-kke 26 529609 58 edk-d(10)-kke 26 529634 49 kde-d(10)-kke 26 529659 52 kddk-d(9)-kke 26 529684 48 kdde-d(9)-kke 26 529709 61 eddk-d(9)-kke 26 529922 52 eeee-d(9)-kke 26 529344 50 eek-d(10)-keke 285 529138 32 eee-d(10)-kkk 286 529158 75 kkk-d(10)-eee 286 529184 75 eek-d(10)-keke 215 529221 78 eek-d(10)-kkke 215 529127 67 eee-d(10)-kkk 287 529147 79 kkk-d(10)-eee 287 529346 58 eek-d(10)-keke 288 529348 65 eek-d(10)-keke 289 529350 77 eek-d(10)-keke 290 529813 20 k-d(10)-kekee 291 529832 47 k-d(10)-kdkee 291 529734 63 ke-d(10)-keke 292 529754 58 ek-d(10)-keke 292 529774 49 ke-d(10)-kdke 292 529794 51 ek-d(10)-kdke 292 529863 64 kde-d(10)-kdke 293 529883 78 edk-d(10)-kdke 293 529903 36 k-d(4)-k-d(4)-k-d(4)-ke 293 529560 71 eek-d(10)-kke 27 529585 70 kee-d(10)-kke 27 529610 66 edk-d(10)-kke 27 529635 45 kde-d(10)-kke 27 529660 53 kddk-d(9)-kke 27 529685 42 kdde-d(9)-kke 27 529710 60 eddk-d(9)-kke 27 529923 63 eeee-d(9)-kke 27 529196 74 eek-d(10)-keke 294 529233 80 eek-d(10)-kkke 294 529139 75 eee-d(10)-kkk 295 529159 62 kkk-d(10)-eee 295 529352 74 eek-d(10)-keke 296 529354 67 eek-d(10)-keke 297 529197 43 eek-d(10)-keke 298 529234 58 eek-d(10)-kkke 298 529140 29 eee-d(10)-kkk 299 529160 59 kkk-d(10)-eee 299 529180 80 eek-d(10)-keke 216 529217 79 eek-d(10)-kkke 216 529814 51 k-d(10)-kekee 300 529833 52 k-d(10)-kdkee 300 529735 43 ke-d(10)-keke 301 529755 60 ek-d(10)-keke 301 529775 38 ke-d(10)-kdke 301 529795 58 ek-d(10)-kdke 301 529864 41 kde-d(10)-kdke 302 529884 48 edk-d(10)-kdke 302 529904 44 k-d(4)-k-(d4)-k-d(4)-ke 302 529934 61 eek-d(10)-keke 302 529356 71 eek-d(10)-keke 303 529561 75 eek-d(10)-kke 28 529586 65 kee-d(10)-kke 28 529611 54 edk-d(10)-kke 28 529636 39 kde-d(10)-kke 28 529661 67 kddk-d(9)-kke 28 529686 66 kdde-d(9)-kke 28 529711 60 eddk-d(9)-kke 28 529924 62 eeee-d(9)-kke 28 529358 82 eek-d(10)-keke 304 529181 79 eek-d(10)-keke 217 529218 73 eek-d(10)-kkke 217 529182 85 eek-d(10)-keke 218 529219 84 eek-d(10)-kkke 218 529360 84 eek-d(10)-keke 305 529362 87 eek-d(10)-keke 306 529364 81 eek-d(10)-keke 307 529366 77 eek-d(10)-keke 308 529198 28 eek-d(10)-keke 309 529235 8 eek-d(10)-kkke 309 529141 34 eee-d(10)-kkk 310 529161 66 kkk-d(10)-eee 310 529368 27 eek-d(10)-keke 311 529370 44 eek-d(10)-keke 312 529372 61 eek-d(10)-keke 313 529374 71 eek-d(10)-keke 314 529376 63 eek-d(10)-keke 315 529378 68 eek-d(10)-keke 316 529380 79 eek-d(10)-keke 317 529382 77 eek-d(10)-keke 318 529384 75 eek-d(10)-keke 319 529386 40 eek-d(10)-keke 320 529240 73 eek-d(10)-keke 321 529241 67 eek-d(10)-keke 322 529242 42 eek-d(10)-keke 323 529243 60 eek-d(10)-keke 324 529388 65 eek-d(10)-keke 325 529815 37 k-d(10)-kekee 326 529834 44 k-d(10)-kdkee 326 529736 47 ke-d(10)-keke 327 529756 78 ek-d(10)-keke 327 529776 37 ke-d(10)-kdke 327 529796 71 ek-d(10)-kdke 327 529865 70 kde-d(10)-kdke 328 529885 59 edk-d(10)-kdke 328 529905 54 k-(d4)-k-(d4)-k-(d4)-ke 328 529935 70 eek-d(10)-keke 328 529562 87 eek-d(10)-kke 29 529587 68 kee-d(10)-kke 29 529612 67 edk-d(10)-kke 29 529637 64 kde-d(10)-kke 29 529662 62 kddk-d(9)-kke 29 529687 63 kdde-d(9)-kke 29 529712 61 eddk-d(9)-kke 29 529925 61 eeee-d(9)-kke 29 529816 77 k-d(10)-kekee 329 529835 80 k-d(10)-kdkee 329 529737 82 ke-d(10)-keke 330 529757 83 ek-d(10)-keke 330 529777 68 ke-d(10)-kdke 330 529797 77 ek-d(10)-kdke 330 529866 15 kde-d(10)-kdke 331 529886 71 edk-d(10)-kdke 331 529906 63 k-(d4)-k-(d4)-k-(d4)-ke 331 529936 78 eek-d(10)-keke 331 529563 89 eek-d(10)-kke 30 529588 84 kee-d(10)-kke 30 529613 80 edk-d(10)-kke 30 529638 48 kde-d(10)-kke 30 529663 85 kddk-d(9)-kke 30 529688 42 kdde-d(9)-kke 30 529713 81 eddk-d(9)-kke 30 529926 67 eeee-d(9)-kke 30 529390 53 eek-d(10)-keke 332 529392 63 eek-d(10)-keke 333 529394 58 eek-d(10)-keke 334 529396 56 eek-d(10)-keke 335 529398 62 eek-d(10)-keke 336 529400 44 eek-d(10)-keke 337 529402 39 eek-d(10)-keke 338 529404 46 eek-d(10)-keke 339 529406 63 eek-d(10)-keke 340 529244 58 eek-d(10)-keke 341 529245 68 eek-d(10)-keke 342 529246 60 eek-d(10)-keke 343 529247 36 eek-d(10)-keke 344 529248 43 eek-d(10)-keke 345 529249 23 eek-d(10)-keke 346 529250 69 eek-d(10)-keke 347 529251 15 eek-d(10)-keke 348 529252 44 eek-d(10)-keke 349 529253 42 cck-d(10)-keke 350 529408 67 eek-d(10)-keke 351 529410 19 eek-d(10)-keke 352 529412 57 eek-d(10)-keke 353 529414 80 eek-d(10)-keke 354 529416 85 eek-d(10)-keke 355 529418 70 eek-d(10)-keke 356 529420 78 eek-d(10)-keke 357 529422 19 eek-d(10)-keke 358 529424 48 eek-d(10)-keke 359 529426 66 eek-d(10)-keke 360 529428 59 eek-d(10)-keke 361 529430 83 eek-d(10)-keke 362 529432 84 eek-d(10)-keke 363 529199 71 eek-d(10)-keke 364 529236 76 eek-d(10)-kkke 364 529142 64 eee-d(10)-kkk 365 529162 60 kkk-d(10)-eee 365 529254 46 eek-d(10)-keke 366 529255 52 eek-d(10)-keke 367 529256 57 eek-d(10)-keke 368 529257 55 eek-d(10)-keke 369 529258 3 eek-d(10)-keke 370 529259 71 eek-d(10)-keke 371 529260 72 eek-d(10)-keke 372 529261 56 eek-d(10)-keke 373 529262 56 eek-d(10)-keke 374 529263 59 eek-d(10)-keke 375 529264 49 eek-d(10)-keke 376 529434 83 eek-d(10)-keke 377 529436 80 eek-d(10)-keke 378 529438 79 eek-d(10)-keke 379 529440 87 eek-d(10)-keke 380 579447 6R eek-d(10)-keke 381 529443 72 eek-d(10)-keke 382 529444 68 eek-d(10)-keke 383 529445 85 eek-d(10)-keke 384 529446 72 eek-d(10)-keke 385 529447 60 eek-d(10)-keke 386 529448 77 eek-d(10)-keke 387 529807 78 k-d(10)-kekee 388 529826 61 k-d(10)-kdkee 388 529449 81 eek-d(10)-keke 389 529728 75 kc-d(10)-kekc 390 529748 80 ek-d(10)-keke 390 529768 68 ke-d(10)-kdke 390 529788 74 ek-d(10)-kdke 390 529857 67 kde-d(10)-kdke 389 529877 77 edk-d(10)-kdke 389 529897 26 k-(d4)-k-(d4)-k-(d4)-ke 389 529200 78 eek-d(10)-keke 391 529237 84 eek-d(10)-kkke 391 529564 90 eek-d(10)-kke 34 529589 86 kee-d(10)-kke 34 529614 82 edk-d(10)-kke 34 529639 80 kde-d(10)-kke 34 529664 69 kddk-d(9)-kke 34 529689 71 kdde-d(9)-kke 34 529714 73 eddk-d(9)-kke 34 529917 73 eeee-d(9)-kke 34 529143 68 eee-d(10)-kkk 392 529163 50 kkk-d(10)-eee 392 529201 76 eek-d(10)-keke 393 529238 72 eek-d(10)-kkke 393 529144 57 eee-d(10)-kkk 394 529164 71 kkk-d(10)-eee 394 529450 91 eek-d(10)-keke 395 529451 85 eek-d(10)-keke 396 529266 63 eek-d(10)-keke 397 529806 52 k-d(10)-kekee 398 529825 44 k-d(10)-kdkee 398 529267 56 eek-d(10)-keke 399 529727 67 ke-d(10)-keke 400 529747 63 ek-d(10)-keke 400 529767 67 ke-d(10)-kdke 400 529787 68 ek-d(10)-kdke 400 529856 42 kde-d(10)-kdke 399 529876 36 edk-d(10)-kdke 399 529896 56 k-(d4)-k-(d4)-k-(d4)-ke 399 529546 65 eek-d(10)-kke 248 529571 80 kee-d(10)-kke 248 529596 43 edk-d(10)-kke 248 529621 38 kde-d(10)-kke 248 529646 68 kddk-d(9)-kke 248 529671 50 kdde-d(9)-kke 248 529696 53 eddk-d(9)-kke 248 529916 22 eeee-d(9)-kke 248 529547 86 eek-d(10)-kke 37 529572 75 kee-d(10)-kke 37 529597 58 edk-d(10)-kke 37 529622 58 kde-d(10)-kke 37 529647 18 kddk-d(9)-kke 37 529672 23 kdde-d(9)-kke 37 529697 28 eddk-d(9)-kke 37 529928 36 eeee-d(9)-kke 37 529452 63 eek-d(10)-keke 401 529453 73 eek-d(10)-keke 402 529454 82 eek-d(10)-keke 403 529455 84 eek-d(10)-keke 404 529202 61 eek-d(10)-keke 405 529239 59 eek-d(10)-kkke 405 529145 54 eee-d(10)-kkk 406 529165 77 kkk-d(10)-eee 406 529456 69 eek-d(10)-keke 407 529457 81 eek-d(10)-keke 408 529458 72 eek-d(10)-keke 409 529459 86 eek-d(10)-keke 410 529460 88 eek-d(10)-keke 411 529817 46 k-d(10)-kekee 412 529836 49 k-d(10)-kdkee 412 529738 51 ke-d(10)-keke 413 529758 53 ek-d(10)-keke 413 529778 39 ke-d(10)-kdke 413 529798 52 ek-d(10)-kdke 413 529867 56 kde-d(10)-kdke 414 529887 68 edk-d(10)-kdke 414 529907 28 k-(d4)-k-(d4)-k-(d4)-ke 414 529938 64 eek-d(10)-keke 414 529565 81 eek-d(10)-kke 38 529590 49 kee-d(10)-kke 38 529615 65 edk-d(10)-kke 38 529640 54 kde-d(10)-kke 38 529665 77 kddk-d(9)-kke 38 529690 77 kdde-d(9)-kke 38 529715 63 eddk-d(9)-kke 38 529927 62 eeee-d(9)-kke 38 529185 66 eek-d(10)-keke 221 529222 62 eek-d(10)-kkke 221 529808 75 k-d(10)-kekee 89 529827 67 k-d(10)-kdkee 89 529128 64 eee-d(10)-kkk 415 529148 78 kkk-d(10)-eee 415 529461 87 eek-d(10)-keke 416 529729 71 ke-d(10)-keke 415 529749 83 ek-d(10)-keke 415 529769 63 ke-d(10)-kdke 415 529789 10 ek-d(10)-kdke 415 529800 69 k-d(10)-kekee 415 529819 78 k-d(10)-kdkee 415 529858 60 kde-d(10)-kdke 416 529878 75 edk-d(10)-kdke 416 529898 34 k-(d4)-k-(d4)-k-(d4)-ke 416 529566 61 eek-d(10)-kke 39 529591 71 kee-d(10)-kke 39 529616 71 edk-d(10)-kke 39 529641 65 kde-d(10)-kke 39 529666 70 kddk-d(9)-kke 39 529691 67 kdde-d(9)-kke 39 529716 75 eddk-d(9)-kke 39 529721 71 ke-d(10)-keke 39 529741 81 ek-d(10)-keke 39 529761 66 ke-d(10)-kdke 39 529781 65 ek-d(10)-kdke 39 529801 71 k-d(10)-kekee 39 529820 74 k-d(10)-kdkee 39 529850 63 kde-d(10)-kdke 417 529870 72 edk-d(10)-kdke 417 529890 23 k-(d4)-k-(d4)-k-(d4)-ke 417 529918 54 eeee-d(9)-kke 39 529567 75 eek-d(10)-kke 262 529592 80 kee-d(10)-kke 262 529617 65 edk-d(10)-kke 262 529642 62 kde-d(10)-kke 262 529667 75 kddk-d(9)-kke 262 529692 53 kdde-d(9)-kke 262 529717 69 eddk-d(9)-kke 262 529722 74 ke-d(10)-keke 262 529742 81 ek-d(10)-keke 262 529762 66 ke-d(10)-kdke 262 529782 68 ek-d(10)-kdke 262 529851 68 kde-d(10)-kdke 418 529871 77 edk-d(10)-kdke 418 529891 36 k-(d4)-k-(d4)-k-(d4)-ke 418 529910 60 eeee-d(9)-kke 262 529568 79 eek-d(10)-kke 263 529593 70 kee-d(10)-kke 263 529618 77 edk-d(10)-kke 263 529643 72 kde-d(10)-kke 263 529668 73 kddk-d(9)-kke 263 529693 62 kdde-d(9)-kke 263 529718 69 eddk-d(9)-kke 263 529911 66 eeee-d(9)-kke 263 529462 76 eek-d(10)-keke 419 529268 18 eek-d(10)-keke 420 529187 46 eek-d(10)-keke 421 529224 48 eek-d(10)-kkke 421 529130 34 eee-d(10)-kkk 422 529150 51 kkk-d(10)-eee 422 529549 85 eek-d(10)-kke 42 529574 81 kee-d(10)-kke 42 529599 64 edk-d(10)-kke 42 529624 68 kde-d(10)-kke 42 529649 77 kddk-d(9)-kke 42 529674 65 kdde-d(9)-kke 42 529699 63 eddk-d(9)-kke 42 529931 59 eeee-d(9)-kke 42 529810 80 k-d(10)-kekee 423 529829 67 k-d(10)-kdkee 423 529269 65 eek-d(10)-keke 424 529731 66 ke-d(10)-keke 425 529751 76 ek-d(10)-keke 425 529771 73 ke-d(10)-kdke 425 529791 65 ek-d(10)-kdke 425 529860 73 kde-d(10)-kdke 424 529880 74 edk-d(10)-kdke 424 529900 62 k-(d4)-k-(d4)-k-(d4)-ke 424 529270 69 eek-d(10)-keke 480 529550 81 eek-d(10)-kke 44 529575 88 kee-d(10)-kke 44 529600 78 edk-d(10)-kke 44 529625 74 kde-d(10)-kke 44 529650 81 kddk-d(9)-kke 44 529675 76 kdde-d(9)-kke 44 529700 73 eddk-d(9)-kke 44 529920 67 eeee-d(9)-kke 44 529271 43 eek-d(10)-keke 427 529272 0 eek-d(10)-keke 428 529273 62 eek-d(10)-keke 429 529274 78 eek-d(10)-keke 430 529275 70 eek-d(10)-keke 431 529276 73 eek-d(10)-keke 432 529277 71 eek-d(10)-keke 433 529278 72 eek-d(10)-keke 434 529279 10 eek-d(10)-keke 435 529280 11 eek-d(10)-keke 436 529281 82 eek-d(10)-keke 437 529282 87 eek-d(10)-keke 438 529803 71 k-d(10)-kekee 250 529822 72 k-d(10)-kdkee 250 529724 76 ke-d(10)-keke 439 529744 81 ek-d(10)-keke 439 529764 65 ke-d(10)-kdke 439 529784 68 ek-d(10)-kdke 439 529853 64 kde-d(10)-kdke 440 529873 69 edk-d(10)-kdke 440 529893 45 k-(d4)-k-(d4)-k-(d4)-ke 440 529937 81 eek-d(10)-keke 440 529551 88 eek-d(10)-kke 48 529576 71 kee-d(10)-kke 48 529601 74 edk-d(10)-kke 48 529626 72 kde-d(10)-kke 48 529651 85 kddk-d(9)-kke 48 529676 67 kdde-d(9)-kke 48 529701 82 eddk-d(9)-kke 48 529913 76 eeee-d(9)-kke 48 529811 56 k-d(10)-kekee 441 529830 46 k-d(10)-kdkee 441 529732 63 ke-d(10)-keke 442 529752 72 ek-d(10)-keke 442 529772 61 ke-d(10)-kdke 442 529792 68 ek-d(10)-kdke 442 529861 54 kde-d(10)-kdke 443 529881 78 edk-d(10)-kdke 443 529901 29 k-(d4)-k-(d4)-k-(d4)-ke 443 529939 67 eek-d(10)-keke 443 529283 70 eek-d(10)-keke 444 529552 72 eek-d(10)-kke 49 529577 80 kee-d(10)-kke 49 529602 64 edk-d(10)-kke 49 529627 56 kde-d(10)-kke 49 529652 57 kddk-d(9)-kke 49 529677 43 kdde-d(9)-kke 49 529702 54 eddk-d(9)-kke 49 529921 42 eeee-d(9)-kke 49 529284 76 eek-d(10)-keke 445 529285 77 eek-d(10)-keke 446 529286 68 eek-d(10)-keke 447 529287 65 eek-d(10)-keke 448 529719 73 ke-d(10)-keke 264 529739 83 ek-d(10)-keke 264 529759 63 ke-d(10)-kdke 264 529779 70 ek-d(10)-kdke 244 529848 60 kde-d(10)-kdke 449 529868 63 edk-d(10)-kdke 449 529888 53 k-(d4)-k-(d4)-k-(d4)-ke 449 529553 81 eek-d(10)-kke 265 529578 65 kee-d(10)-kke 265 529603 60 edk-d(10)-kke 265 529628 59 kde-d(10)-kke 265 529653 76 kddk-d(9)-kke 265 529678 56 kdde-d(9)-kke 265 529703 68 eddk-d(9)-kke 265 529908 69 eeee-d(9)-kke 265 529168 64 eek-d(10)-keke 450 529205 62 eek-d(10)-kkke 450 529290 53 eek-d(10)-keke 451 529802 57 k-d(10)-kekee 452 529821 61 k-d(10)-kdkee 452 529292 74 eek-d(10)-keke 453 529723 68 ke-d(10)-keke 454 529743 84 ek-d(10)-keke 454 529763 64 ke-d(10)-kdke 454 529783 72 ek-d(10)-kdke 454 529852 66 kde-d(10)-kdke 453 529872 62 edk-d(10)-kdke 453 529892 43 k-(d4)-k-(d4)-k-(d4)-ke 453 529554 80 eek-d(10)-kke 252 529579 83 kee-d(10)-kke 252 529604 73 edk-d(10)-kke 252 529629 64 kde-d(10)-kke 252 529654 69 kddk-d(9)-kke 252 529679 52 kddc-d(9)-kkc 252 529704 63 eddk-d(9)-kke 252 529912 64 eeee-d(9)-kke 252 529294 74 eek-d(10)-keke 455 529296 52 eek-d(10)-keke 456 529298 60 eek-d(10)-keke 457 529300 71 eek-d(10)-keke 458 529188 79 eek-d(10)-keke 459 529225 78 eek-d(10)-kkke 459 529131 58 eee-d(10)-1dck 460 529151 71 kkk-d(10)-eee 460 529302 74 eek-d(10)-keke 461 529189 64 eek-d(10)-keke 222 529226 50 eek-d(10)-kkke 222 529132 78 eee-d(10)-kkk 462 529152 62 kkk-d(10)-eee 462 529190 76 eek-d(10)-keke 223 529227 88 eek-d(10)-kkke 250 529133 81 eee-d(10)-kkk 463 529153 68 kkk-d(10)-eee 463 529191 78 eek-d(10)-keke 224 529228 85 eek-d(10)-kkke 224 529134 75 eee-d(10)-kkk 464 529154 61 kkk-d(10)-eee 464 529304 89 eek-d(10)-keke 465 529306 84 eek-d(10)-keke 466 529308 68 eek-d(10)-keke 467 529310 59 eek-d(10)-keke 468 .529169 79 eek-d(10)-keke 469 529206 82 eek-d(10)-kkke 469 529312 68 eek-d(10)-keke 470 529314 61 eek-d(10)-keke 471 529316 62 eek-d(10)-keke 472 529555 78 eek-d(10)-kke 59 529580 73 kee-d(10)-kke 59 529605 71 edk-d(10)-kke 59 529630 64 kde-d(10)-kke 59 529655 63 kddk-d(9)-kke 59 529680 43 kdde-d(9)-kke 59 529705 63 eddk-d(9)-kke 59 529932 60 eeee-d(9)-kke 59 529318 82 eek-d(10)-keke 473 529170 85 eek-d(10)-keke 474 529207 88 eek-d(10)-kkke 474 529171 81 eek-d(10)-keke 475 529208 84 eek-d(10)-kkke 475 529805 40 k-d(10)-kekee 476 529824 32 k-d(10)-kdkee 476 529320 74 eek-d(10)-keke 477 529726 80 ke-d(10)-keke 478 529746 82 ek-d(10)-keke 478 529766 63 ke-d(10)-kdke 478 529786 69 ek-d(10)-kdke 478 529855 39 kde-d(10)-kdke 477 529875 40 edk-d(10)-kdke 477 529895 27 k-(d4)-k-(d4)-k-(d4)-ke 477 529556 72 eek-d(10)-kke 61 529581 68 kee-d(10)-kke 61 529606 54 edk-d(10)-kke 61 529631 29 kde-d(10)-kke 61 529656 74 kddk-d(9)-kke 61 529681 32 kdde-d(9)-kke 61 529706 41 eddk-d(9)-kke 61 529915 51 eeee-d(9)-kke 61 529172 88 eek-d(10)-keke 226 529209 87 eek-d(10)-kkke 226 529173 92 eek-d(10)-keke 227 529210 89 eek-d(10)-kkke 227 529183 85 eek-d(10)-keke 479 529220 92 eek-d(10)-kkke 479 529126 83 eee-d(10)-kkk 257 529146 84 kkk-d(10)-eee 257 529174 85 eek-d(10)-keke 480 529211 86 eek-d(10)-kkke 480 529322 71 eek-d(10)-keke 481 529324 79 eek-d(10)-keke 482 529326 85 ee.k-d(10)-keke 4R3 529175 92 eek-d(10)-keke 228 529212 92 eek-d(10)-kkke 228 529176 89 eek-d(10)-keke 229 529213 90 eek-d(10)-kkke 229 529804 89 k-d(10)-kekee 259 529823 89 k-d(10)-kdkee 259 529166 83 eek-d(10)-keke 230 529203 86 eek-d(10)-kkke 230 529725 92 ke-d(10)-keke 260 529745 91 ek-d(10)-keke 260 529765 88 ke-d(10)-kdke 260 529785 91 ek-d(10)-kdke 260 529799 89 k-d(10)-kekee 260 529818 88 k-d(10)-kdkee 260 529854 90 kde-d(10)-kdke 230 529874 81 edk-d(10)-kdke 230 529894 60 k-(d4)-k-(d4)-k-(d4)-ke 230 529167 71 eek-d(10)-keke 231 529204 70 eek-d(10)-kkke 231 529557 86 eek-d(10)-kke 69 529582 86 kee-d(10)-kke 69 529607 84 edk-d(10)-kke 69 529632 81 kde-d(10)-kke 69 529657 85 kddk-d(9)-kke 69 529682 78 kdde-d(9)-kke 69 529707 79 eddk-d(9)-kke 69 529720 75 ke-d(10)-keke 69 529740 70 ek-d(10)-keke 69 529760 78 ke-d(10)-kdke 69 529780 83 ek-d(10)-kdke 69 529849 80 kde-d(10)-kdke 231 529869 72 edk-d(10)-kdke 231 529889 49 k-(d4)-k-(d4)-k-(d4)-ke 231 529914 69 eeee-d(9)-kke 69 529328 68 eek-d(10)-keke 484 529558 71 eek-d(10)-kke 71 529583 81 kee-d(10)-kke 71 529608 68 edk-d(10)-kke 71 529633 73 kde-d(10)-kke 71 529658 63 kddk-d(9)-kke 71 529683 74 kdde-d(9)-kke 71 529708 70 eddk-d(9)-kke 71 529909 59 eeee-d(9)-kke 71 529192 51 eek-d(10)-keke 485 529229 69 eek-d(10)-kkke 485 529135 54 eee-d(10)-kkk 486 529155 56 kkk-d(10)-eee 486 529330 37 eek-d(10)-keke 487 e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

Example 20 Design of Modified Oligonucleotides Comprising 2′-O-methoxyethyl (2′-MOE) or Constrained Ethyl (cEt) Modifications

Based on the activity of the antisense oligonucleotides listed above, additional antisense oligonucleotides were designed targeting a Target-X nucleic acid targeting start positions 1147, 1154 or 12842 of Target-X

The newly designed chimeric antisense oligonucleotides are 16 or 17 nucleotides in length and their motifs are described in Table 31. The chemistry column of Table 31 presents the sugar motif of each oligonucleotide, wherein “e” indicates a 2′-O-methythoxylethyl (2′-MOE) nucleoside, “k” indicates a constrained ethyl (cEt) nucleoside and “d” indicates a 2′-deoxyribonucleoside. The internucleoside linkages throughout each gapmer are hosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosine.

Each gapmer listed in Table 31 is targeted to the human Target-X genomic sequence.

TABLE 31 Chimeric antisense oligonucleotides targeted to Target-X ISIS No Chemistry SEQ CODE 529544 eek-d(10)-kke 21 529569 kee-d(10)-kke 21 529594 edk-d(10)-kke 21 529619 kde-d(10)-kke 21 529644 kddk-d(9)-kke 21 529669 kdde-d(9)-kke 21 529694 eddk-d(9)-kke 21 529929 eeee-d(9)-kke 21 529809 k-d(10)-kekee 488 529828 k-d(10)-kdkee 488 529730 ke-d(10)-keke 489 529750 ek-d(10)-keke 489 579770 ke-d(10)-kdke 489 529790 ek-d(10)-kdke 489 529859 kde-d(10)-kdke 490 529879 edk-d(10)-kdke 490 529899 k-d(4)-k-d(4)-k-d(4)-ke 490 529545 eek-d(10)-kke 22 529570 kee-d(10)-kke 22 529595 edk-d(10)-kke 22 529620 kde-d(10)-kke 22 529645 kddk-d(9)-kke 22 529670 kdde-d(9)-kke 22 529695 eddk-d(9)-kke 22 529919 eeee-d(9)-kke 22 529548 eek-d(10)-kke 41 529573 kee-d(10)-kke 41 529598 edk-d(10)-kke 41 529623 kde-d(10)-kke 41 529648 kddk-d(9)-kke 41 529673 kdde-d(9)-kke 41 529698 eddk-d(9)-kke 41 529930 eeee-d(9)-kke 41 e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

Example 21 Modified Oligonucleotides Comprising 2′-O-methoxyethyl (2′-MOE) and Constrained Ethyl (cEt) Modifications Targeting Human Target-X

Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. ISIS 472998 and ISIS 515554, described in the Examples above were also included in the screen.

The newly designed chimeric antisense oligonucleotides are 16 nucleotides in length and their motifs are described in Table 32. The chemistry column of Table 32 presents the sugar motif of each oligonucleotide, wherein “e” indicates a 2′-O-methythoxylethyl (2′-MOE) nucleoside, “k” indicates a constrained ethyl (cEt) nucleoside and “d” indicates a 2′-deoxyribonucleoside. The internucleoside linkages throughout each gapmer are hosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.

Each gapmer listed in Table 32 is targeted to the human Target-X genomic sequence.

Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells.

TABLE 32 Inhibition of human Target-X mRNA levels by chimeric antisense oligonucleotides targeted to Target-X ISIS No % inhibition Chemistry SEQ CODE 472998 88 kk-d(10)-kk 74 515554 75 eee-d(10)-kkk 493 534530 92 keke-d(9)-kek 491 534563 92 kek-d(9)-ekek 491 534596 88 ekee-d(9)-kke 491 534629 89 eke-d(9)-ekke 491 534662 87 eekk-d(9)-eke 491 534695 92 eek-d(9)-keke 491 534732 90 ekek-d(8)-keke 491 534767 92 keek-d(8)-keek 491 534802 93 ekk-d(10)-kke 491 534832 83 edk-d(10)-kke 491 534862 72 kde-d(10)-kke 491 534892 82 eek-d(10)-kke 491 534922 80 kddk-d(9)-kke 491 534952 72 kdde-d(9)-kke 491 534982 77 eddk-d(9)-kke 491 535012 70 eeee-d(9)-kke 491 535045 84 eeee-d(9)-kkk 491 535078 87 eeek-d(9)-kke 491 535111 63 eeeee-d(8)-kke 491 535144 69 ededk-d(8)-kke 491 535177 68 edkde-d(8)-kke 491 534531 61 keke-d(9)-kek 492 534564 30 kek-d(9)-ekek 492 534597 67 ekee-d(9)-kke 492 534630 54 eke-d(9)-ekke 492 534663 94 eekk-d(9)-eke 492 534696 68 eek-d(9)-keke 492 534733 44 ekek-d(8)-keke 492 534768 55 keek-d(8)-keek 492 534803 73 ekk-d(10)-kke 492 534833 65 edk-d(10)-kke 492 534863 53 kde-d(10)-kke 492 534893 61 eek-d(10)-kke 492 534923 70 kddk-d(9)-kke 492 534953 54 kdde-d(9)-kke 492 534983 58 eddk-d(9)-kke 492 535013 52 eeee-d(9)-kke 492 535046 67 eeee-d(9)-kkk 492 535079 57 eeek-d(9)-kke 492 535112 42 eeeee-d(8)-kke 492 535145 41 ededk-d(8)-kke 492 535178 35 edkde-d(8)-kke 492 534565 87 kek-d(9)-ekek 493 534598 72 ekee-d(9)-kke 493 534631 70 eke-d(9)-ekke 493 534664 94 eekk-d(9)-eke 493 534697 90 eek-d(9)-keke 493 534734 74 ekek-d(8)-keke 493 534769 80 keek-d(8)-keek 493 534804 87 ekk-d(10)-kke 493 534834 76 edk-d(10)-kke 493 534864 56 kde-d(10)-kke 493 534894 67 eek-d(10)-kke 493 534924 71 kddk-d(9)-kke 493 534954 54 kdde-d(9)-kke 493 534984 48 eddk-d(9)-kke 493 535014 43 eeee-d(9)-kke 493 535047 60 eeee-d(9)-kkk 493 535080 64 eeek-d(9)-kke 493 535113 32 eeeee-d(8)-kke 493 535146 31 ededk-d(8)-kke 493 535179 28 edkde-d(8)-kke 493 534533 82 keke-d(9)-kek 494 534566 88 kek-d(9)-ekek 494 534599 65 ekee-d(9)-kke 494 534632 69 eke-d(9)-ekke 494 534665 87 eekk-d(9)-eke 494 534698 64 eek-d(9)-keke 494 534735 63 ekek-d(8)-keke 494 534770 66 keek-d(8)-keek 494 534805 87 ekk-d(10)-kke 494 534835 68 edk-d(10)-kke 494 514865 66 kde-d(10)-kke 494 534895 57 eek-d(10)-kke 494 534925 82 kddk-d(9)-kke 494 534955 76 kdde-d(9)-kke 494 534985 71 eddk-d(9)-kke 494 535015 59 eeee-d(9)-kke 494 535048 69 eeee-d(9)-kkk 494 535081 67 eeek-d(9)-kke 494 535114 37 eeeee-d(8)-kke 494 535147 32 ededk-d(8)-kke 494 535180 31 edkde-d(8)-kke 494 534534 94 keke-d(9)-kek 234 534567 92 kek-d(9)-ekek 234 534600 92 ekee-d(9)-kke 234 534633 91 eke-d(9)-ekke 234 534666 89 eekk-d(9)-eke 234 534699 91 eek-d(9)-keke 234 534736 83 ekek-d(8)-keke 234 534771 80 keek-d(8)-keek 234 534806 96 ekk-d(10)-kke 234 534836 86 edk-d(10)-kke 234 534866 82 kde-d(10)-kke 234 534896 82 eek-d(10)-kke 234 534926 89 kddk-d(9)-kke 234 534956 91 kdde-d(9)-kke 234 534986 87 eddk-d(9)-kke 234 535016 83 eeee-d(9)-kke 234 535049 87 eeee-d(9)-kkk 234 535082 87 eeek-d(9)-kke 234 535115 77 eeeee-d(8)-kke 234 535148 73 ededk-d(8)-kke 234 535181 68 edkde-d(8)-kke 234 534535 66 keke-d(9)-kek 236 534568 85 kek-d(9)-ekek 236 534601 51 ekee-d(9)-kke 236 534634 80 eke-d(9)-ekke 236 534667 90 eekk-d(9)-eke 236 534700 88 eek-d(9)-keke 236 534737 65 ekek-d(8)-keke 236 534772 77 keek-d(8)-keek 236 534807 84 ekk-d(10)-kke 236 534837 78 edk-d(10)-kke 236 534867 44 kde-d(10)-kke 236 534R97 82 eek-d(10)-kke 236 534927 61 kddk-d(9)-kke 236 534957 58 kdde-d(9)-kke 236 534987 49 eddk-d(9)-kke 236 535017 38 eeee-d(9)-kke 236 535050 32 eeee-d(9)-kkk 236 535083 43 eeek-d(9)-kke 236 535116 9 eeeee-d(8)-kke 236 535149 23 ededk-d(8)-kke 236 535182 18 edkde-d(8)-kke 236 534536 89 keke-d(9)-kek 238 534569 90 kek-d(9)-ekek 238 534602 85 ekee-d(9)-kke 238 534635 87 eke-d(9)-ekke 238 534668 90 eekk-d(9)-eke 238 534701 92 eek-d(9)-keke 238 534738 81 ekek-d(8)-keke 238 534773 79 keek-d(8)-keek 238 534808 90 ekk-d(10)-kke 238 534838 88 edk-d(10)-kke 238 534868 67 kde-d(10)-kke 238 534898 89 eek-d(10)-kke 238 534928 81 kddk-d(9)-kke 238 534958 78 kdde-d(9)-kke 238 534988 66 eddk-d(9)-kke 238 535018 78 eeee-d(9)-kke 238 535051 76 eeee-d(9)-kkk 238 535084 80 eeek-d(9)-kke 238 535117 58 eeeee-d(8)-kke 238 535150 51 ededk-d(8)-kke 238 535183 53 edkde-d(8)-kke 238 534537 91 keke-d(9)-kek 239 534570 85 kek-d(9)-ekek 239 534603 79 ekee-d(9)-kke 239 534636 72 eke-d(9)-ekke 239 534669 85 eekk-d(9)-eke 239 534702 85 eek-d(9)-keke 239 534739 73 ekek-d(8)-keke 239 534774 77 keek-d(8)-keek 239 534809 91 ekk-d(10)-kke 239 534839 86 edk-d(10)-kke 239 534869 71 kde-d(10)-kke 239 534899 82 eek-d(10)-kke 239 534929 83 kddk-d(9)-kke 239 534959 80 kdde-d(9)-kke 239 534989 79 eddk-d(9)-kke 239 535019 76 eeee-d(9)-kke 239 535052 79 eeee-d(9)-kkk 239 535085 81 eeek-d(9)-kke 239 535118 58 eeeee-d(8)-kke 239 535151 65 ededk-d(8)-kke 239 535184 60 edkde-d(8)-kke 239 534516 77 keke-d(9)-kek 495 534549 80 kek-d(9)-ekek 495 534582 73 ekee-d(9)-kke 495 534615 79 eke-d(9)-ekke 495 534648 67 eekk-d(9)-eke 495 534681 87 eek-d(9)-keke 495 534718 46 ekek-d(8)-keke 495 534753 68 keek-d(8)-keek 495 534788 84 ekk-d(10)-kke 495 534818 82 edk-d(10)-kke 495 534848 75 kde-d(10)-kke 495 534878 72 eek-d(10)-kke 495 534908 81 kddk-d(9)-kke 495 534938 69 kdde-d(9)-kke 495 534968 77 eddk-d(9)-kke 495 534998 76 eeee-d(9)-kke 495 535031 76 eeee-d(9)-kkk 495 535064 70 eeek-d(9)-kke 495 535097 57 eeeee-d(8)-kke 495 535130 69 ededk-d(8)-kke 495 535163 58 edkde-d(8)-kke 495 534538 71 keke-d(9)-kek 241 534571 64 kek-d(9)-ekek 241 534604 66 ekee-d(9)-kke 241 534637 74 eke-d(9)-ekke 241 534670 87 eekk-d(9)-eke 241 534703 72 eek-d(9)-keke 241 534740 56 ekek-d(8)-keke 241 534775 53 keek-d(8)-keek 241 534810 78 ekk-d(10)-kke 241 534840 73 edk-d(10)-kke 241 534870 65 kde-d(10)-kke 241 534900 69 eek-d(10)-kke 241 534930 67 kddk-d(9)-kke 241 534960 62 kdde-d(9)-kke 241 534990 66 eddk-d(9)-kke 241 535020 61 eeee-d(9)-kke 241 535053 47 eeee-d(9)-kkk 241 535086 61 eeek-d(9)-kke 241 535119 49 eeeee-d(8)-kke 241 535152 48 ededk-d(8)-kke 241 535185 57 edkde-d(8)-kke 241 534539 70 keke-d(9)-kek 496 534572 82 kek-d(9)-ekek 496 534605 59 ekee-d(9)-kke 496 534638 69 eke-d(9)-ekke 496 534671 89 eekk-d(9)-eke 496 534704 83 eek-d(9)-keke 496 534741 47 ekek-d(8)-keke 496 534776 46 keek-d(8)-keek 496 534811 71 ekk-d(10)-kke 496 534841 61 edk-d(10)-kke 496 534871 53 kde-d(10)-kke 496 534901 55 eek-d(10)-kke 496 534931 73 kddk-d(9)-kke 496 534961 53 kdde-d(9)-kke 496 534991 56 eddk-d(9)-kke 496 535021 58 eeee-d(9)-kke 496 535054 59 eeee-d(9)-kkk 496 535087 0 eeek-d(9)-kke 496 535120 41 eeeee-d(8)-kke 496 535153 44 ededk-d(8)-kke 496 535186 35 edkde-d(8)-kke 496 534573 76 kek-d(9)-ekek 497 534606 55 ekee-d(9)-kke 497 534639 72 eke-d(9)-ekke 497 534672 89 eekk-d(9)-eke 497 534705 87 eek-d(9)-keke 497 534742 84 ekek-d(8)-keke 497 534777 79 keek-d(8)-keek 497 534812 76 ekk-d(10)-kke 497 534842 74 edk-d(10)-kke 497 534872 53 kde-d(10)-kke 497 534902 70 eek-d(10)-kke 497 534932 73 kddk-d(9)-kke 497 534962 60 kdde-d(9)-kke 497 534992 61 eddk-d(9)-kke 497 535022 38 eeee-d(9)-kke 497 535055 42 eeee-d(9)-kkk 497 535088 56 eeek-d(9)-kke 497 535121 5 eeeee-d(8)-kke 497 535154 22 ededk-d(8)-kke 497 535187 16 edkde-d(8)-kke 497 534541 86 keke-d(9)-kek 498 534574 89 kek-d(9)-ekek 498 534607 59 ekee-d(9)-kke 498 534640 76 eke-d(9)-ekke 498 534673 89 eekk-d(9)-eke 498 534706 86 eek-d(9)-keke 498 534743 79 ekek-d(8)-keke 498 534778 80 keek-d(8)-keek 498 534813 83 ekk-d(10)-kke 498 534843 82 edk-d(10)-kke 498 534873 83 kde-d(10)-kke 498 534903 78 eek-d(10)-kke 498 534933 83 kddk-d(9)-kke 498 534963 70 kdde-d(9)-kke 498 534993 78 eddk-d(9)-kke 498 535023 56 eeee-d(9)-kke 498 535056 59 eeee-d(9)-kkk 498 535089 73 eeek-d(9)-kke 498 535122 39 eeeee-d(8)-kke 498 535155 60 ededk-d(8)-kke 498 535188 41 edkde-d(8)-kke 498 534542 75 keke-d(9)-kek 499 534575 82 kek-d(9)-ekek 499 534608 72 ekee-d(9)-kke 499 534641 69 eke-d(9)-ekke 499 534674 84 eekk-d(9)-eke 499 534707 78 eek-d(9)-keke 499 534744 72 ekek-d(8)-keke 499 534779 75 keek-d(8)-keek 499 534814 81 ekk-d(10)-kke 499 534844 75 edk-d(10)-kke 499 534874 70 kde-d(10)-kke 499 534904 71 eek-d(10)-kke 499 534934 73 kddk-d(9)-kke 499 534964 72 kdde-d(9)-kke 499 534994 69 eddk-d(9)-kke 499 535024 56 eeee-d(9)-kke 499 535057 63 eeee-d(9)-kkk 499 535090 64 eeek-d(9)-kke 499 535123 40 eeeee-d(8)-kke 499 535156 47 ededk-d(8)-kke 499 535189 48 edkde-d(8)-kke 499 534515 52 keke-d(9)-kek 34 534548 85 kek-d(9)-ekek 34 534581 75 ekee-d(9)-kke 34 534614 83 eke-d(9)-ekke 34 534647 65 eekk-d(9)-eke 34 534680 88 eek-d(9)-keke 34 534717 76 ekek-d(8)-keke 34 534752 79 keek-d(8)-keek 34 534787 90 ekk-d(10)-kke 34 535030 77 eeee-d(9)-kkk 34 535063 75 eeek-d(9)-kke 34 535096 54 eeeee-d(8)-kke 34 535129 66 ededk-d(8)-kke 34 535162 49 edkde-d(8)-kke 34 534543 66 keke-d(9)-kek 500 534576 69 kek-d(9)-ekek 500 534609 77 ekee-d(9)-kke 500 534642 62 eke-d(9)-ekke 500 534675 80 eekk-d(9)-eke 500 534708 81 eek-d(9)-keke 500 534745 68 ekek-d(8)-keke 500 534780 69 keek-d(8)-keek 500 534815 85 ekk-d(10)-kke 500 534845 72 edk-d(10)-kke 500 534875 56 kde-d(10)-kke 500 534905 65 eek-d(10)-kke 500 534935 78 kddk-d(9)-kke 500 534965 48 kdde-d(9)-kke 500 534995 62 eddk-d(9)-kke 500 535025 58 eeee-d(9)-kke 500 535058 60 eeee-d(9)-kkk 500 535091 61 eeek-d(9)-kke 500 535124 51 eeeee-d(8)-kke 500 535157 55 ededk-d(8)-kke 500 535190 47 edkde-d(8)-kke 500 534517 71 keke-d(9)-kek 501 534550 80 kek-d(9)-ekek 501 534583 70 ekee-d(9)-kke 501 514616 84 eke-d(9)-ekke 501 534649 68 eekk-d(9)-eke 501 534682 87 eek-d(9)-keke 501 534719 90 ekek-d(8)-keke 501 534754 83 keek-d(8)-keek 501 534789 86 ekk-d(10)-kke 501 534819 69 edk-d(10)-kke 501 534849 62 kde-d(10)-kke 501 534879 69 eek-d(10)-kke 501 534909 73 kddk-d(9)-kke 501 534939 49 kdde-d(9)-kke 501 534969 47 eddk-d(9)-kke 501 534999 51 eeee-d(9)-kke 501 535032 51 eeee-d(9)-kkk 501 535065 64 eeek-d(9)-kke 501 535098 31 eeeee-d(8)-kke 501 535131 31 ededk-d(8)-kke 501 535164 40 edkde-d(8)-kke 501 534518 81 keke-d(9)-kek 502 534551 88 kek-d(9)-ekek 502 534584 78 ekee-d(9)-kke 502 534617 80 eke-d(9)-ekke 502 534650 83 eekk-d(9)-eke 502 534683 93 eek-d(9)-keke 502 534720 87 ekek-d(8)-keke 502 534755 82 keek-d(8)-keek 502 534790 89 ekk-d(10)-kke 502 534820 64 edk-d(10)-kke 502 534850 38 kde-d(10)-kke 502 534880 68 eek-d(10)-kke 502 534910 60 kddk-d(9)-kke 502 534940 37 kdde-d(9)-kke 502 534970 59 eddk-d(9)-kke 502 535000 30 eeee-d(9)-kke 502 535033 44 eeee-d(9)-kkk 502 535066 64 eeek-d(9)-kke 502 535099 22 eeeee-d(8)-kke 502 535132 54 ededk-d(8)-kke 502 535165 45 edkde-d(8)-kke 502 534544 80 keke-d(9)-kek 503 534577 83 kek-d(9)-ekek 503 534610 62 ekee-d(9)-kke 503 534643 66 eke-d(9)-ekke 503 534676 95 eekk-d(9)-eke 503 534709 86 eek-d(9)-keke 503 534746 73 ekek-d(8)-keke 503 534781 71 keek-d(8)-keek 503 534816 83 ekk-d(10)-kke 503 534846 73 edk-d(10)-kke 503 534876 39 kde-d(10)-kke 503 534906 67 eek-d(10)-kke 503 534936 66 kddk-d(9)-kke 503 534966 48 kdde-d(9)-kke 503 534996 56 eddk-d(9)-kke 503 535026 39 eeee-d(9)-kke 503 535059 45 eeee-d(9)-kkk 503 535092 48 eeek-d(9)-kke 503 535125 26 eeeee-d(8)-kke 503 535158 44 ededk-d(8)-kke 503 535191 34 edkde-d(8)-kke 503 534545 83 keke-d(9)-kek 504 534578 81 kek-d(9)-ekek 504 534611 78 ekee-d(9)-kke 504 534644 72 eke-d(9)-ekke 504 534677 92 eekk-d(9)-eke 504 534710 78 eek-d(9)-keke 504 534747 85 ekek-d(8)-keke 504 534782 85 keek-d(8)-keek 504 534817 88 ekk-d(10)-kke 504 534847 73 edk-d(10)-kke 504 534877 66 kde-d(10)-kke 504 534907 73 eek-d(10)-kke 504 534937 85 kddk-d(9)-kke 504 534967 80 kdde-d(9)-kke 504 534997 74 eddk-d(9)-kke 504 535027 64 eeee-d(9)-kke 504 535060 68 eeee-d(9)-kkk 504 535093 73 eeek-d(9)-kke 504 535126 42 eeeee-d(8)-kke 504 535159 49 ededk-d(8)-kke 504 535192 51 edkde-d(8)-kke 504 534519 87 keke-d(9)-kek 505 534552 85 kek-d(9)-ekek 505 534585 76 ekee-d(9)-kke 505 534618 78 eke-d(9)-ekke 505 534651 79 eekk-d(9)-eke 505 534684 87 eek-d(9)-keke 505 534721 89 ekek-d(8)-keke 505 534756 90 keek-d(8)-keek 505 534791 84 ekk-d(10)-kke 505 534821 79 edk-d(10)-kke 505 534851 64 kde-d(10)-kke 505 534881 65 eek-d(10)-kke 505 534911 85 kddk-d(9)-kke 505 534941 66 kdde-d(9)-kke 505 534971 75 eddk-d(9)-kke 505 535001 62 eeee-d(9)-kke 505 535034 65 eeee-d(9)-kkk 505 535067 76 eeek-d(9)-kke 505 535100 5 eeeee-d(8)-kke 505 535133 30 ededk-d(8)-kke 505 535166 23 edkde-d(8)-kke 505 534520 87 keke-d(9)-kek 251 534553 79 kek-d(9)-ekek 251 534586 60 ekee-d(9)-kke 251 534619 62 eke-d(9)-ekke 251 534652 84 eekk-d(9)-eke 251 534685 84 eek-d(9)-keke 251 534722 75 ekek-d(8)-keke 251 534757 81 keek-d(8)-keek 251 534792 87 ekk-d(10)-kke 251 534822 80 edk-d(10)-kke 251 534852 38 kde-d(10)-kke 251 534882 75 eek-d(10)-kke 251 534912 74 kddk-d(9)-kke 251 534942 58 kdde-d(9)-kke 251 534972 59 eddk-d(9)-kke 251 535002 50 eeee-d(9)-kke 251 535035 57 eeee-d(9)-kkk 251 535068 67 eeek-d(9)-kke 251 535101 24 eeeee-d(8)-kke 251 535134 23 ededk-d(8)-kke 251 535167 26 edkde-d(8)-kke 251 534513 90 keke-d(9)-kek 252 534546 92 kek-d(9)-ekek 252 534579 78 ekee-d(9)-kke 252 534612 82 eke-d(9)-ekke 252 534645 73 eekk-d(9)-eke 252 534678 91 eek-d(9)-keke 252 514715 87 ekek-d(8)-keke 252 534750 88 keek-d(8)-keek 252 534785 89 ekk-d(10)-kke 252 535028 52 eeee-d(9)-kkk 252 535061 73 eeek-d(9)-kke 252 535094 61 eeeee-d(8)-kke 252 535127 59 ededk-d(8)-kke 252 535160 62 edkde-d(8)-kke 252 534521 86 keke-d(9)-kek 506 534554 87 kek-d(9)-ekek 506 534587 62 ekee-d(9)-kke 506 534620 68 eke-d(9)-ekke 506 534653 77 eekk-d(9)-eke 506 534686 90 eek-d(9)-keke 506 534723 88 ekek-d(8)-keke 506 534758 79 keek-d(8)-keek 506 534793 85 ekk-d(10)-kke 506 534823 81 edk-d(10)-kke 506 534853 59 kde-d(10)-kke 506 534883 69 eek-d(10)-kke 506 534913 76 kddk-d(9)-kke 506 534943 53 kdde-d(9)-kke 506 534973 61 eddk-d(9)-kke 506 535003 53 eeee-d(9)-kke 506 535036 35 eeee-d(9)-kkk 506 535069 62 eeek-d(9)-kke 506 535102 31 eeeee-d(8)-kke 506 535135 44 ededk-d(8)-kke 506 535168 34 edkde-d(8)-kke 506 534522 83 keke-d(9)-kek 507 534555 81 kek-d(9)-ekek 507 534588 72 ekee-d(9)-kke 507 534621 74 eke-d(9)-ekke 507 534654 78 eekk-d(9)-eke 507 534687 91 eek-d(9)-keke 507 534724 84 ekek-d(8)-keke 507 534759 86 keek-d(8)-keek 507 534794 78 ekk-d(10)-kke 507 534824 75 edk-d(10)-kke 507 534854 63 kde-d(10)-kke 507 534884 60 eek-d(10)-kke 507 534914 75 kddk-d(9)-kke 507 534944 69 kdde-d(9)-kke 507 534974 66 eddk-d(9)-kke 507 535004 56 eeee-d(9)-kke 507 535037 50 eeee-d(9)-kkk 507 535070 68 eeek-d(9)-kke 507 535103 55 eeeee-d(8)-kke 507 535136 51 ededk-d(8)-kke 507 535169 54 edkde-d(8)-kke 507 534523 89 keke-d(9)-kek 253 534556 91 kek-d(9)-ekek 253 534589 88 ekee-d(9)-kke 253 534622 93 eke-d(9)-ekke 253 534655 72 eekk-d(9)-eke 253 534688 92 eek-d(9)-keke 253 534725 87 ekek-d(8)-keke 253 534760 92 keek-d(8)-keek 253 534795 93 ekk-d(10)-kke 253 534825 82 edk-d(10)-kke 253 534855 73 kde-d(10)-kke 253 534885 82 eek-d(10)-kke 253 534915 88 kddk-d(9)-kke 253 534945 82 kdde-d(9)-kke 253 534975 68 eddk-d(9)-kke 253 535005 69 eeee-d(9)-kke 253 535038 72 eeee-d(9)-kkk 253 535071 74 eeek-d(9)-kke 253 535104 61 eeeee-d(8)-kke 253 535137 67 ededk-d(8)-kke 253 535170 51 edkde-d(8)-kke 253 534524 95 keke-d(9)-kek 254 534557 98 kek-d(9)-ekek 254 534590 91 ekee-d(9)-kke 254 534623 91 eke-d(9)-ekke 254 534656 90 eekk-d(9)-eke 254 534689 92 eek-d(9)-keke 254 534726 57 ekek-d(8)-keke 254 534761 89 keek-d(8)-keek 254 534796 93 ekk-d(10)-kke 254 534826 89 edk-d(10)-kke 254 534856 87 kde-d(10)-kke 254 534886 85 eek-d(10)-kke 254 534916 87 kddk-d(9)-kke 254 534946 86 kdde-d(9)-kke 254 534976 77 eddk-d(9)-kke 254 535006 83 eeee-d(9)-kke 254 535039 86 eeee-d(9)-kkk 254 535072 87 eeek-d(9)-kke 254 535105 68 eeeee-d(8)-kke 254 535138 70 ededk-d(8)-kke 254 535171 65 edkde-d(8)-kke 254 534558 92 kek-d(9)-ekek 255 534591 91 ekee-d(9)-kke 255 534624 86 eke-d(9)-ekke 255 534657 90 eekk-d(9)-eke 255 534690 76 eek-d(9)-keke 255 534727 92 ekek-d(8)-keke 255 534762 91 keek-d(8)-keek 255 534797 94 ekk-d(10)-kke 255 534827 90 edk-d(10)-kke 255 534857 80 kde-d(10)-kke 255 534887 76 eek-d(10)-kke 255 534917 91 kddk-d(9)-kke 255 534947 91 kdde-d(9)-kke 255 534977 86 eddk-d(9)-kke 255 535007 80 eeee-d(9)-kke 255 535040 86 eeee-d(9)-kkk 255 535073 87 eeek-d(9)-kke 255 535106 70 eeeee-d(8)-kke 255 535139 73 ededk-d(8)-kke 255 535172 69 edkde-d(8)-kke 255 534514 90 keke-d(9)-kek 61 534547 92 kek-d(9)-ekek 61 534580 78 ekee-d(9)-kke 61 534613 80 eke-d(9)-ekke 61 534646 79 eekk-d(9)-eke 61 534679 93 eek-d(9)-keke 61 534716 94 ekek-d(8)-keke 61 534751 86 keek-d(8)-keek 61 534786 83 ekk-d(10)-kke 61 535029 45 eeee-d(9)-kkk 61 535062 81 eeek-d(9)-kke 61 535095 57 eeeee-d(8)-kke 61 535128 58 ededk-d(8)-kke 61 535161 49 edkde-d(8)-kke 61 534526 94 keke-d(9)-kek 256 534559 95 kek-d(9)-ekek 256 534592 93 ekee-d(9)-kke 256 514625 93 eke-d(9)-ekke 256 534658 93 eekk-d(9)-eke 256 534691 96 eek-d(9)-keke 256 534728 93 ekek-d(8)-keke 256 534763 93 keek-d(8)-keek 256 534798 97 ekk-d(10)-kke 256 534828 94 edk-d(10)-kke 256 534858 92 kde-d(10)-kke 256 534888 93 eek-d(10)-kke 256 534918 95 kddk-d(9)-kke 256 534948 93 kdde-d(9)-kke 256 534978 91 eddk-d(9)-kke 256 535008 88 eeee-d(9)-kke 256 535041 87 eeee-d(9)-kkk 256 535074 90 eeek-d(9)-kke 256 535107 78 eeeee-d(8)-kke 256 535140 81 ededk-d(8)-kke 256 535173 81 edkde-d(8)-kke 256 534527 95 keke-d(9)-kek 258 -534560 96 kek-d(9)-ekek 258 534593 87 ekee-d(9)-kke 258 534626 85 eke-d(9)-ekke 258 534659 90 eekk-d(9)-eke 258 534692 91 eek-d(9)-keke 258 534729 91 ekek-d(8)-keke 258 534764 91 keek-d(8)-keek 258 534799 96 ekk-d(10)-kke 258 534829 91 edk-d(10)-kke 258 534859 87 kde-d(10)-kke 258 534889 81 eek-d(10)-kke 258 534919 92 kddk-d(9)-kke 258 534949 91 kdde-d(9)-kke 258 534979 84 eddk-d(9)-kke 258 535009 78 eeee-d(9)-kke 258 535042 76 eeee-d(9)-kkk 258 535075 83 eeek-d(9)-kke 258 535108 64 eeeee-d(8)-kke 258 535141 69 ededk-d(8)-kke 258 535174 65 edkde-d(8)-kke 258 534528 94 keke-d(9)-kek 260 534561 0 kek-d(9)-ekek 260 534594 92 ekee-d(9)-kke 260 534627 90 eke-d(9)-ekke 260 534660 92 eekk-d(9)-eke 260 534693 95 eek-d(9)-keke 260 534730 93 ekek-d(8)-keke 260 534765 92 keek-d(8)-keek 260 534800 93 ekk-d(10)-kke 260 534830 93 edk-d(10)-kke 260 534860 85 kde-d(10)-kke 260 534890 91 eek-d(10)-kke 260 534920 93 kddk-d(9)-kke 260 534950 90 kdde-d(9)-kke 260 534980 88 eddk-d(9)-kke 260 535010 88 eeee-d(9)-kke 260 535043 89 eeee-d(9)-kkk 260 535076 88 eeek-d(9)-kke 260 535109 76 eeeee-d(8)-kke 260 535142 86 ededk-d(8)-kke 260 535175 71 edkde-d(8)-kke 260 534529 70 keke-d(9)-kek 261 534562 86 kek-d(9)-ekek 261 534595 56 ekee-d(9)-kke 261 534628 73 eke-d(9)-ekke 261 534661 64 eekk-d(9)-eke 261 534694 75 eek-d(9)-keke 261 534731 47 ekek-d(8)-keke 261 534766 30 keek-d(8)-keek 261 534801 83 ekk-d(10)-kke 261 534831 84 edk-d(10)-kke 261 534861 71 kde-d(10)-kke 261 534891 73 eek-d(10)-kke 261 534921 55 kddk-d(9)-kke 261 534951 61 kdde-d(9)-kke 261 534981 48 eddk-d(9)-kke 261 535011 54 eeee-d(9)-kke 261 535044 46 eeee-d(9)-kkk 261 535077 29 eeek-d(9)-kke 261 535110 19 eeeee-d(8)-kke 261 535143 15 ededk-d(8)-kke 261 535176 37 edkde-d(8)-kke 261 e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

Example 22 Modified Antisense Oligonucleotides Comprising 2′-O-methoxyethyl (2′-MOE) and Constrained Ethyl (cEt) Modifications Targeting Human Target-X Targeting Intronic Repeats

Additional antisense oligonucleotides were designed targeting the intronic repeat regions of Target-X

The newly designed chimeric antisense oligonucleotides and their motifs are described in Table 33. The internucleoside linkages throughout each gapmer are phosphorothioate linkages (P═S) and are designated as “s”. Nucleosides followed by “d” indicate 2′-deoxyribonucleosides. Nucleosides followed by “k” indicate constrained ethyl (cEt) nucleosides. Nucleosides followed by “e” indicate 2′-O-methythoxylethyl (2′-MOE) nucleosides. “N” indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).

Each gapmer listed in Table 33 is targeted to the intronic region of human Target-X genomic sequence, designated herein as Target-X.

Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells.

TABLE 33 Inhibition of human Target-X mRNA levels by chimeric antisense oligonucleotides targetedt o Target-X ISIS % in- SEQ SEQ ID Sequence (5′ to 3′) No hibition CODE NO Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds 472998 90 508 20 Nds Nks Nk Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds 473327 88  30 19 Nds Nds Nes Nes Ne Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537024 74 509 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537025 79 510 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537026 76 511 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537028 37 512 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537029 45 513 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537030 67 514 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537031 59 515 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537032  9 516 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537033 65 517 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537034 71 518 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537035 68 519 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537036 74 520 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537038 69 521 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537039 67 522 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537040 68 523 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537041 76 524 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537042 77 525 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537043 70 526 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537044 82 527 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537045 69 528 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537047 35 529 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537049 62 530 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537051 62 531 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537055 16 532 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537056 25 533 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537057 49 534 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537058 49 535 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537059 53 536 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537060 73 537 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537061 70 538 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537062 69 539 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537063 68 540 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537064 71 541 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537065 67 542 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537066 68 543 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537067 71 544 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537068 86 545 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537069 82 546 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537070 87 547 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537792 36 548 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537793 35 549 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537794 35 550 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537795 33 551 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537796 49 552 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537797 54 553 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537798 68 554 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537799 72 555 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537800 69 556 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537801 82 557 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537802 72 558 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537803 72 559 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537804 67 560 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537805 74 561 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537806 70 562 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537809 60 563 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537810 71 564 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537811 69 565 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537812 80 566 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537813 74 567 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537814 54 568 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537837 70 569 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537838 76 570 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537839 76 571 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537840 80 572 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537841 81 573 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537842 75 574 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537843 70 575 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537844 73 576 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537845 59 577 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537846 51 578 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537847 52 579 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537848 41 580 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537849 44 581 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538160 69 582 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538172 24 583 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538173 23 584 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538185 68 585 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538187 69 585 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538189 81 587 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538191 66 588 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538192 59 589 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538193 16 590 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538194 10 591 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538195 15 592 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538196  3 593 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538197 36 594 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538198 49 595 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538199 47 596 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538200 57 597 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538201 71 598 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538202 60 599 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538203 55 600 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538204 62 601 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538205 68 602 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538228 63 603 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538229 26 604 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538230 75 605 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538231 75 606 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538233 52 607 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538235 26 608 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538237 28 609 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538239 54 610 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538241 73 611 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538242 68 612 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538243 61 613 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538245 75 614 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538253 37 615 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538254 45 616 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538361 56 617 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538378 70 618 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538380 68 619 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538381 57 620 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540361 71 621 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540362 73 622 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540363 78 623 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540364 89 624 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540365 83 625 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540366 84 626 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540367 65 627 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540368 55 628 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540369 82 629 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540370 86 630 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540371 74 631 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540372 82 632 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540373 81 633 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540374 87 634 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540375 78 635 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540376 69 636 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540377 88 637 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540378 85 638 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540379 77 639 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540380 84 640 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540381 85 641 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540382 69 642 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540383 85 643 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540384 88 644 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540385 87 645 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540386 86 646 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540387 77 647 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540388 86 648 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540389 86 649 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540390 85 650 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540391 83 651 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540392 43 652 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540393 88 653 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540394 68 654 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540395 87 655 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540396 87 656 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540397 59 657 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540398 36 658 19 Nds Nds Nks Nks Nk Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540399 81 659 19 Nds Nds Nks Nks Nk

Example 23 High Dose Tolerability of Modified Oligonucleotides Comprising 2′-O-methoxyethyl (2′-MOE) and 6′-(S)—CH3 Bicycle Nucleoside (e.g cEt) Modifications Targeting Human Target-X in BALB/c Mice

BALB/c mice were treated at a high dose with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.

Additionally, the newly designed antisense oligonucleotides were created with the same sequences as the antisense oligonucleotides from the study described above and were also added to this screen targeting intronic repeat regions of Target-X.

The newly designed modified antisense oligonucleotides and their motifs are described in Table 34. The internucleoside linkages throughout each gapmer are phosphorothioate linkages (P═S). Nucleosides followed by “d” indicate 2′-deoxyribonucleosides. Nucleosides followed by “k” indicate 6′-(S)—CH3 bicyclic nucleoside (e.g cEt) nucleosides. Nucleosides followed by “e” indicate 2′-O-methythoxylethyl (2′-MOE) nucleosides. “N” indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).

Each gapmer listed in Table 34 is targeted to the intronic region of human Target-X genomic sequence, designated herein as Target-X.

TABLE 34 Modified antisense oligonucleotides targeted to Target-X ISIS SEQ SEQ ID Sequence (5 to 3′) No CODE NO Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537721 509 19 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537738 524 19 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537759 539 19 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537761 541 19 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537763 543 19 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537850 548 19 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537858 556 19 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537864 562 19 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537869 565 19 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537872 568 19 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537897 571 19 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 540118 582 19 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 540138 602 19 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 540139 603 19 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 540148 612 19 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 540153 617 19 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 540155 619 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540162 624 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540164 626 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540168 630 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540172 634 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540175 637 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540176 638 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540178 640 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540179 641 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540181 643 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540182 644 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540183 645 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540184 646 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540186 648 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540187 649 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540188 650 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540191 653 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540193 655 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540194 656 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544811 547 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544812 545 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544813 527 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544814 557 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544815 546 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544816 573 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544817 572 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544818 566 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544819 510 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544820 525 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544821 567 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544826 537 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544827 538 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544828 539 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544829 540 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544830 541 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545471 542 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545472 543 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545473 544 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545474 558 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545475 559 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545476 560 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545477 561 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545478 562 19 Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545479 556 19 Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537727 514 19

Treatment

Male BALB/c mice were injected subcutaneously with a single dose of 200 mg/kg of ISIS 422142, ISIS 457851, ISIS 473294, ISIS 473295, ISIS 473327, ISIS 484714, ISIS 515334, ISIS 515338, ISIS 515354, ISIS 515366, ISIS 515380, ISIS 515381, ISIS 515382, ISIS 515384, ISIS 515386, ISIS 515387, ISIS 515388, ISIS 515406, ISIS 515407, ISIS 515408, ISIS 515422, ISIS 515423, ISIS 515424, ISIS 515532, ISIS 515533, ISIS 515534, ISIS 515538, ISIS 515539, ISIS 515558, ISIS 515656, ISIS 515575, ISIS 515926, ISIS 515944, ISIS 515945, ISIS 515948, ISIS 515949, ISIS 515951, ISIS 515952, ISSI 516003, ISIS 516055, ISIS 516057, ISIS 516060, ISIS 516062, ISIS 529126, ISIS 529146, ISIS 529166, ISIS 529170, ISIS 529172, ISIS 529173, ISIS 529174, ISIS 529175, ISSI 529176, ISIS 529182, ISIS 529183, ISIS 529186, ISIS 529282, ISIS 529304, ISIS 529306, ISIS 529360, ISIS 529450, ISIS 529459, ISIS 529460, ISIS 529461, ISIS 529547, ISIS 529550, ISIS 529551, ISIS 529553, ISIS 529557, ISIS 529562, ISIS 529563, ISIS 529564, ISIS 529565, ISIS 529575, ISIS 529582, ISIS 529589, ISIS 529607, ISIS 529614, ISIS 529632, ISIS 529650, ISIS 529651, ISIS 529657, ISIS 529663, ISIS 529725, ISIS 529745, ISIS 529765, ISIS 529785, ISIS 529804, ISIS 529818, ISIS 529823, ISIS 529854, ISIS 534528, ISIS 534534, ISIS 534594, ISIS 534660, ISIS 534663, ISIS 534664, ISIS 534676, ISIS 534677, ISIS 537679, ISIS 537683, ISIS 534693, ISIS 534701, ISIS 534716, ISIS 534730, ISIS 534765, ISIS 534795, ISIS 534796, ISIS 534797, ISIS 534798, ISIS 534799, ISIS 534800, ISIS 534802, ISIS 534806, ISSI 534830, ISIS 534838, ISIS 534888, ISIS 534890, ISIS 534898, ISIS 534911, ISIS 534920, ISIS 534926, ISIS 534937, ISIS 534950, ISSI 534956, ISIS 534980, ISIS 534986, ISIS 535010, ISIS 535043, ISIS 535049, ISIS 535076, ISIS 535082, ISSI 535142, ISIS 537024, ISIS 537030, ISIS 537041, ISIS 537062, ISIS 537064, ISIS 537066, ISIS 537721, ISIS 537727, ISIS 537738, ISIS 537759, ISIS 537761, ISIS 537763, ISIS 537792, ISIS 537800, ISIS 537806, ISIS 537811, ISIS 537814, ISIS 537839, ISIS 537850, ISSI 537858, ISIS 537864, ISIS 537869, ISIS 537872, ISIS 537897, ISIS 538160, ISIS 538196, ISIS 538205, ISIS 538228, ISIS 538242, ISIS 538361, ISIS 538380, ISIS 540118, ISIS 540138, ISIS 540139, ISIS 540148, ISIS 540153, ISIS 540155, ISIS 540162, ISIS 540164, ISIS 540168, ISIS 540172, ISIS 540175, ISIS 540176, ISIS 540178, ISIS 540179, ISIS 540181, ISIS 540182, ISIS 540183, ISIS 540184, ISIS 540186, ISIS 540187, ISIS 540188, ISIS 540191, ISIS 540193, ISIS 540194, ISIS 544811, ISIS 544812, ISIS 544813, ISIS 544814, ISIS 544815, ISIS 544816, ISIS 544817, ISIS 544818, ISIS 544819, ISIS 544820, ISIS 544821, ISIS 544826, ISIS 544827, ISIS 544828, ISIS 544829, ISIS 544830, ISIS 545471, ISIS 545472, ISIS 545473, ISIS 545474, ISIS 545475, ISIS 545476, ISIS 545477, ISIS 545478, and ISIS 545479. One set of male BALB/c mice was injected with a single dose of PBS. Mice were euthanized 96 hours later, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).

ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 529166, ISIS 529170, ISIS 529175, ISIS 529176, ISIS 529186, ISIS 529282, ISIS 529360, ISIS 529450, ISIS 529459, ISIS 529460, ISIS 529547, ISIS 529549, ISIS 529551, ISIS 529553, ISIS 529557, ISIS 529562, ISIS 529575, ISIS 529582, ISIS 529607, ISIS 529589, ISIS 529632, ISIS 529657, ISIS 529725, ISIS 529745, ISIS 529785, ISIS 529799, ISIS 529804, ISIS 529818, ISIS 529823, ISIS 534950, ISIS 534980, ISIS 535010, ISIS 537030, ISIS 537041, ISIS 537062, ISIS 537064, ISIS 537066, ISIS 537759, ISIS 537792, ISIS 537800, ISIS 537839, ISIS 538228, ISIS 473294, ISIS 473295, ISIS 484714, ISIS 515338, ISIS 515366, ISIS 515380, ISIS 515381, ISIS 515387, ISIS 515408, ISIS 515423, ISIS 515424, ISIS 515532, ISIS 515534, ISIS 515538, ISIS 515539, ISIS 515558, ISIS 515575, ISIS 515926, ISIS 515944, ISIS 515945, ISIS 515951, ISIS 515952, ISIS 529126, ISIS 529765, ISIS 534528, ISIS 534534, ISIS 534594, ISIS 534663, ISIS 534676, ISIS 534677, ISIS 534679, ISIS 534683, ISIS 534693, ISIS 534701, ISIS 534716, ISIS 534730, ISIS 534806, ISIS 534830, ISIS 534838, ISIS 534890, ISIS 534898, ISIS 534911, ISIS 534937, ISIS 534956, ISIS 534986, ISIS 535043, ISIS 535049, ISIS 535076, ISIS 535082, ISIS 535142, ISIS 538160, ISIS 538242, ISIS 538361, ISIS 538380, ISIS 534795, ISIS 534796, ISIS 534797, ISIS 540162, ISIS 540164, ISIS 540168, ISIS 540172, ISIS 540175, ISIS 540176, ISIS 540178, ISIS 540179, ISIS 540181, ISIS 540182, ISIS 540183, ISIS 540184, ISIS 540186, ISIS 540187, ISIS 540188, ISIS 540191, ISIS 540193, ISIS 540194, ISIS 544813, ISIS 544814, ISIS 544816, ISIS 544826, ISIS 544827, ISIS 544828, ISIS 544829, ISIS 545473, and ISIS 545474 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 529173, ISIS 529854, ISIS 529614, ISIS 515386, ISIS 515388, ISIS 515949, ISIS 544817, and ISIS 545479 were considered tolerable in terms of liver function.

Example 24 Tolerability of Antisense Oligonucleotides Targeting Human Target-X in Sprague-Dawley Rats

Sprague-Dawley rats are a multipurpose model used for safety and efficacy evaluations. The rats were treated with ISIS antisense oligonucleotides from the studies described in the Examples above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

Six-eight week old male Sprague-Dawley rats were maintained on a 12-hour light/dark cycle and fed ad libitum with Teklad normal rat chow. Groups of four Sprague-Dawley rats each were injected subcutaneously twice a week for 6 weeks with 25 mg/kg of ISIS 473286, ISIS 473547, ISIS 473567, ISIS 473589, ISIS 473630, ISIS 484559, ISIS 515636, ISIS 515640, ISIS 515641, ISIS 515655, ISIS 515657, ISIS 516046, ISIS 516048, ISIS 516051, ISIS 516052, and ISIS 516062. A group of four Sprague-Dawley rats was injected subcutaneously twice a week for 6 weeks with PBS. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase) were measured. Plasma levels of Bilirubin and BUN were also measured using the same clinical chemistry analyzer. ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 473286, ISIS 473547, ISSI 473589, ISIS 473630, ISIS 484559, ISIS 515636, ISIS 515640, ISIS 515655, ISIS 516046, and ISIS 516051 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 473567, ISIS 515641, ISIS 515657, ISIS 516048, and ISIS 516051 were considered tolerable in terms of liver function.

Example 25 Tolerability of Chimeric Antisense Oligonucleotides Comprising 2′-O-methoxyethyl (2% MOE) Modifications Targeting Human Target-X in Sprague-Dawley Rats

Sprague-Dawley rats were treated with ISIS antisense oligonucleotides from the studies described in the Examples above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

Six-eight week old male Sprague-Dawley rats were maintained on a 12-hour light/dark cycle and fed ad libitum with Purina normal rat chow. Groups of four Sprague-Dawley rats each were injected subcutaneously twice a week for 6 weeks with 50 mg/kg of ISIS 407936, ISIS 416507, ISIS 416508, ISIS 490208, ISIS 490279, ISIS 490323, ISIS 490368, ISIS 490396, ISIS 490803, ISIS 491122, ISIS 513419, ISIS 513446, ISIS 513454, ISIS 513455, ISIS 513456, ISIS 513504, ISIS 513507, and ISIS 513508. A group of four Sprague-Dawley rats was injected subcutaneously twice a week for 6 weeks with PBS. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of Bilirubin and BUN were also measured using the same clinical chemistry analyzer.

ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the

ULN were deemed tolerable. Based on these criteria, ISIS 416507, ISIS 490208, ISIS 490368, ISIS 490396, ISIS 490803, ISIS 491122, ISIS 513446, ISIS 513454, ISIS 513455, ISIS 513456, ISIS 513504, and ISIS 513508 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 407936, ISIS 416508, ISIS 490279, and ISIS 513507 were considered tolerable in terms of liver function.

Example 26 Tolerability of Chimeric Antisense Oligonucleotides Comprising 2′-O-methoxyethyl (2′-MOE) Modifications Targeting Human Target-X in CD-1 Mice

CD-1 mice are a multipurpose mice model, frequently utilized for safety and efficacy testing. The mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

Groups of 3 male CD-1 mice each were injected subcutaneously twice a week for 6 weeks with 50 mg/kg of ISIS 473244, ISIS 473295, ISIS 484714, ISIS 515386, ISIS 515424, ISIS 515534, ISIS 515558, ISIS 515926, ISIS 515949, ISIS 515951, ISIS 515952, ISIS 529126, ISIS 529166, ISIS 529173, ISIS 529186, ISIS 529360, ISIS 529461, ISIS 529553, ISIS 529564, ISIS 529582, ISIS 529614, ISIS 529725, ISIS 529745, ISIS 529765, ISIS 529785, ISIS 529799, ISIS 529818, ISIS 529823, ISIS 534528, ISIS 534594, and ISIS 534664. One group of male CD-1 mice was injected subcutaneously twice a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).

ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 473295, ISIS 473714, ISIS 515558, ISIS 515926, 515951, ISIS 515952, ISIS 529126, ISIS 529166, 529564, ISIS 529582, ISIS 529614, ISIS 529725, ISIS 529765, ISIS 529799, ISIS 529823, and ISIS 534594 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 515424, ISIS 515534, ISIS 515926, ISIS 529785, and ISIS 534664 were considered tolerable in terms of liver function.

Example 27 Tolerability of Chimeric Antisense Oligonucleotides Comprising 2′-O-methoxyethyl (2′-MOE) Modifications Targeting Human Target-X in CD-1 Mice

CD-1 mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

Groups of 3 male CD-1 mice each were injected subcutaneously twice a week for 6 weeks with 100 mg/kg of ISIS 490208, ISIS 490279, ISIS 490323, ISIS 490368, ISIS 490396, ISIS 490803, ISIS 491122, ISIS 513419, ISIS 513446, ISIS 513454, ISIS 513455, ISIS 513456, ISIS 513504, ISIS 513507, and ISIS 513508. Groups of 3 male CD-1 mice each were injected subcutaneously twice a week for 6 weeks with 100 mg/kg of ISIS 407936, ISIS 416507, and ISIS 416508, which are gapmers described in a previous publication. One group of male CD-I mice was injected subcutaneously twice a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).

ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 407936, ISIS 416507, ISIS 490279, ISIS 490368, ISIS 490396, ISIS 490803, ISIS 491122, ISIS 513446, ISIS 513454, ISIS 513456, and ISIS 513504 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 490208, ISIS 513455, ISIS 513507, and ISIS 513508 were considered tolerable in terms of liver function.

Example 28 Efficacy of Modified Oligonucleotides Comprising 2′-O-methoxyethyl (2′-MOE) and Constrained Ethyl (cEt) Modifications Targeting Human Target-X in Transgenic Mice

Transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for efficacy.

Treatment

Groups of 2-3 male and female transgenic mice were injected subcutaneously twice a week for 3 weeks with 5 mg/kg/week of ISIS 473244, ISIS 473295, ISIS 484714, ISIS 515926, ISIS 515951, ISIS 515952, ISIS 516062, ISIS 529126, ISIS 529553, ISIS 529745, ISIS 529799, ISIS 534664, ISIS 534826, ISIS 540168, ISIS 540175, ISIS 544826, ISIS 544827, ISIS 544828, and ISIS 544829. One group of mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Protein Analysis

Plasma protein levels of Target-X were estimated using a Target-X ELISA kit (purchased from Hyphen Bio-Med). Results are presented as percent inhibition of Target-X, relative to control. As shown in Table 35, several antisense oligonucleotides achieved reduction of human Target-X over the PBS control. ‘n.d.’ indicates that the value for that particular oligonucleotide was not measured.

TABLE 35 Percent inhibition of Target-X plasma protein levels in transgenic mice % ISIS No inhibition 473244 2 473295 13 484714 19 515926 11 515951 13 515952 0 516062 62 529126 0 529553 0 529745 22 529799 26 534664 32 534826 n.d. 540168 94 540175 98 544813 0 544826 23 544827 60 544828 33 544829 53

Example 29 Efficacy of Modified Oligonucleotides Comprising 2′-methoxyethyl (2′-MOE) and Constrained Ethyl (cEt) Modifications Targeting Human Target-X in Transgenic Mice

Transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for efficacy.

Treatment

Groups of 2-3 male and female transgenic mice were injected subcutaneously twice a week for 3 weeks with 1 mg/kg/week of ISIS 407936, ISIS 490197, ISIS 490275, ISIS 490278, ISIS 490279, ISIS 490323, ISIS 490368, ISIS 490396, ISIS 490803, ISIS 491122, ISIS 513446, ISIS 513447, ISIS 513504, ISIS 516062, ISIS 529166, ISIS 529173, ISIS 529360, ISIS 529725, ISIS 534557, ISIS 534594, ISIS 534664, ISIS 534688, ISIS 534689, ISIS 534915, ISIS 534916, ISIS 534917, and ISIS 534980. One group of mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Protein Analysis

Plasma protein levels of Target-X were estimated using a Target-X ELISA kit (purchased from Hyphen Bio-Med). Results are presented as percent inhibition of Target-X, relative to control. As shown in Table 36, several antisense oligonucleotides achieved reduction of human Target-X over the PBS control.

TABLE 36 Percent inhibition of Target-X plasm protein levels in transgenic mice % ISIS No inhibition 407936 28 490197 50 490275 21 490278 20 490279 59 490323 54 490368 22 490396 31 490803 30 491122 51 513446 29 513447 44 513504 45 516062 75 529166 37 529173 64 529360 43 529725 53 534557 76 534594 40 534664 14 534687 12 534688 48 534689 25 534915 40 534916 45 534917 66 534980 62

Example 30 Tolerability of Antisense Oligonucleotides Targeting Human Target-X in Sprague-Dawley Rats

Sprague-Dawley rats were treated with ISIS antisense oligonucleotides from the studies described in the Examples above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

Six-eight week old male Sprague-Dawley rats were maintained on a 12-hour light/dark cycle and fed ad libitum with Teklad normal rat chow. Groups of four Sprague-Dawley rats each were injected subcutaneously twice a week for 4 weeks with ISIS 515380, ISIS 515381, ISIS 515387, ISIS 529175, ISIS 529176, ISIS 529575, ISIS 529804, and ISIS 537064. Doses 1, 5, 6, 7, and 8 were 25 mg/kg; dose 2 was 75 mg/kg; doses 3 and 4 were 50 mg/kg. One group of four Sprague-Dawley rats was injected subcutaneously twice a week for 4 weeks with PBS. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases ALT (alanine transaminase) and AST (aspartate transaminase) were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of Bilirubin and BUN were also measured using the same clinical chemistry analyzer.

ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused increase in the levels within three times the upper limit of normal levels of transaminases were deemed very tolerable. ISIS oligonucleotides that caused increase in the levels of transaminases between three times and seven times the upper limit of normal levels were deemed tolerable. Based on these criteria, ISIS 515380, ISIS 515387, ISIS 529175, ISIS 529176, ISIS 529804, and ISIS 537064 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 515381 was considered tolerable in terms of liver function.

Example 31 Efficacy of Antisense Oligonucleotides Targeting Human Target-X in Transgenic Mice

Transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for efficacy.

Treatment

Two groups of 3 male and female transgenic mice were injected subcutaneously twice a week for 2 weeks with 0.5 mg/kg/week or 1.5 mg/kg/week of ISIS 407935 and ISIS 513455. Another group of mice was subcutaneously twice a week for 2 weeks with 0.6 mg/kg/week or 2.0 mg/kg/week of ISIS 473286. Another 16 groups of mice were subcutaneously twice a week for 2 weeks with 0.1 mg/kg/week or 0.3 mg/kg/week of ISIS 473589, ISIS 515380, ISIS 515423, ISIS 529804, ISIS 534676, ISIS 534796, ISIS 540162, ISIS 540164, ISIS 540175, ISIS 540179, ISIS 540181, ISIS 540182, ISIS 540186, ISIS 540191, ISIS 540193, ISIS 544827, or ISIS 545474. Another 3 groups of mice were injected subcutaneously twice a week for 2 weeks with 0.3 mg/kg/week of ISIS 516062, ISIS 534528 or ISIS 534693. One group of mice was injected subcutaneously twice a week for 2 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Protein Analysis

Plasma protein levels of Target-X were estimated using a Target-X ELISA kit (purchased from Hyphen Bio-Med). Results are presented as percent inhibition of Target-X, relative to control. As shown in Table 37, several antisense oligonucleotides achieved reduction of human Target-X over the PBS control.

TABLE 37 Percent inhibition of Target-X plasma protein levels in transgenic mice Dose % ISIS No (mg/kg/wk) inhibition 407935 1.5 65 0.5 31 513455 1.5 64 0.5 52 473286 2 67 0.6 11 473589 0.3 42 0.1 12 515380 0.3 64 0.1 32 515423 0.3 72 0.1 37 529804 0.3 36 0.1 24 534676 0.3 31 0.1 18 534796 0.3 54 0.1 43 540162 0.3 84 0.1 42 540164 0.3 25 0.1 17 540175 0.3 90 0.1 55 540179 0.3 29 0.1 24 540181 0.3 53 0.1 0 540182 0.3 78 0.1 21 540186 0.3 72 0.1 46 540191 0.3 62 0.1 35 540193 0.3 74 0.1 46 544827 0.3 28 0.1 19 545474 0.3 59 0.1 0 516062 0.3 33 534528 0.3 41 534693 0.3 34

Example 32 Tolerability of Antisense Oligonucleotides Targeting Human Target-X in Sprague-Dawley Rats

Sprague-Dawley rats were treated with ISIS antisense oligonucleotides from the studies described in the Examples above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

Five-six week old male Sprague-Dawley rats were maintained on a 12-hour light/dark cycle and fed ad libitum with Teklad normal rat chow. Groups of four Sprague-Dawley rats each were injected subcutaneously twice a week for 4 weeks with 50 mg/kg of ISIS 515423, ISIS 515424, ISIS 515640, ISIS 534676, ISIS 534796, ISIS 534797, ISIS 540162, ISIS 540164, ISIS 540172, ISIS 540175, ISIS 540179, ISIS 540181, ISIS 540182, ISIS 540183, ISIS 540186, ISIS 540191, and ISIS 545474. A group of four Sprague-Dawley rats was injected subcutaneously twice a week for 4 weeks with PBS. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase) were measured. Plasma levels of Bilirubin and BUN were also measured using the same clinical chemistry analyzer.

ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 540164, ISIS 540172, and ISIS 540175 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 534676, ISIS 534796, ISIS 534797, ISIS 540162, and ISIS 540179 were considered tolerable in terms of liver function.

Example 33 Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B Cells

Antisense oligonucleotides selected from the studies described above were tested at various doses in Hep3B cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.05 μM, 0.15 μM, 0.44 μM, 1.33 μM, and 4.00 μM concentrations of antisense oligonucleotide, as specified in Table 38. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human Target-X primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented in Table 38. As illustrated in Table 38, Target-X mRNA levels were reduced in a dose-dependent manner in several of the antisense oligonucleotide treated cells.

TABLE 38 Dose-dependent antisense inhibition of human Target-X in Hep3B cells using electroporation ISIS 0.05 0.15 0.44 1.33 4.00 IC₅₀ No μM μM μM μM μM (μM) 473286 0 1 13 12 15 >4.0 457851 23 32 57 80 93 0.3 473286 3 20 43 71 88 0.5 473286 15 26 24 28 36 >4.0 473286 6 3 10 26 29 >4.0 473327 14 28 35 67 90 0.5 473589 29 53 76 89 95 0.1 515380 44 72 85 93 95 <0.05 515423 43 64 87 95 98 <0.05 515424 38 55 85 92 97 0.1 515636 21 33 74 82 93 0.2 516046 29 23 29 48 78 0.9 516048 35 24 41 67 87 0.4 516052 18 6 48 63 80 0.6 516062 24 14 21 47 68 1.6 529166 16 47 75 87 94 0.2 529173 14 49 77 91 96 0.2 529175 30 69 88 93 96 0.1 529176 34 63 85 93 96 0.1 529360 35 53 74 91 93 0.1 529725 53 69 85 92 95 <0.05 529804 37 41 71 90 94 0.1 534528 50 68 78 93 97 <0.05 534557 48 78 90 94 95 <0.05 534594 39 47 76 87 94 0.1 534676 29 20 40 64 87 0.5 534687 41 37 56 80 93 0.2 534688 16 56 88 94 96 0.1 534689 21 59 82 94 95 0.1 534693 18 58 81 93 95 0.1 534795 19 43 68 90 94 0.2 534796 25 59 80 93 96 0.1 534890 31 55 77 90 96 0.1 534898 22 61 80 94 97 0.1 534915 19 26 51 77 94 0.3 534916 20 36 66 86 93 0.2 534917 34 53 82 89 94 0.1 540162 40 64 84 90 92 <0.05 540164 34 60 83 91 92 0.1 540168 51 79 90 92 94 <0.05 540172 40 66 80 88 92 <0.05 540175 30 61 80 88 91 0.1 540176 7 17 50 75 85 0.5 540179 11 22 25 16 19 >4.0 540181 19 46 72 86 91 0.2 540182 16 66 83 86 92 0.1 540183 39 74 87 92 93 <0.05 540186 31 69 85 91 94 0.1 540191 38 54 80 88 91 0.1 540193 57 67 84 94 97 <0.05 540194 30 45 62 77 91 0.2 544827 37 42 67 82 96 0.1 544829 26 41 42 71 93 0.3 545473 28 27 49 80 97 0.3 545474 23 27 55 84 96 0.3

Example 34 Tolerability of Antisense Oligonucleotides Targeting Human Target-X in CD-1 Mice

CD-1 mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

Two groups of 4 male 6-8 week old CD-1 mice each were injected subcutaneously twice a week for 6 weeks with 50 mg/kg of ISIS 407935 and ISIS 490279. Another seven groups of 4 male 6-8 week old CD-1 mice each were injected subcutaneously twice a week for 6 weeks with 25 mg/kg of ISIS 473589, ISIS 529804, ISIS 534796, ISIS 540162, ISIS 540175, ISIS 540182, and ISIS 540191. One group of male CD-1 mice was injected subcutaneously twice a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in Table 39. Treatment with the newly designed antisense oligonucleotides were more tolerable compared to treatment with ISIS 407935 (disclosed in an earlier publication), which caused elevation of ALT levels greater than seven times the upper limit of normal (ULN).

TABLE 39 Effect of antisense oligonucleotide treatment on liver function in CD-1 mice Dose (mg/kg/ ALT AST BUN Bilirubin Motif wk) (IU/L) (IU/L) (mg/dL) (mg/dL) PBS — — 37 47 28 0.2 407935 e5-d(10)-e5 100 373 217 24 0.2 490279 kdkdk-d(9)-ee 100 96 82 24 0.2 473589 e5-d(10)-e5 50 93 116 22 0.2 529804 k-d(10)-kekee 50 54 74 27 0.2 534796 ekk-d(10)-kke 50 60 63 27 0.2 540162 eek-d(10)-kke 50 43 55 29 0.2 540175 eek-d(10)-kke 50 113 78 24 0.3 540182 eek-d(10)-kke 50 147 95 26 0.1 540191 eek-d(10)-kke 50 79 88 28 0.2 e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside Body and organ weights

Body weights, as well as liver, heart, lungs, spleen and kidney weights were measured at the end of the study, and are presented in Table 40. Several of the ISIS oligonucleotides did not cause any changes in organ weights outside the expected range and were therefore deemed tolerable in terms of organ weight

TABLE 40 Body and organ weights (grams) of CD-1 mice Dose Body Motif (mg/kg/wk) weight Liver Spleen Kidney PBS — — 42 2.2 0.12 0.64 407935 e5-d(10)-e5 100 40 2.6 0.20 0.62 490279 kdkdk-d(9)-ee 100 42 2.8 0.17 0.61 473589 e5-d(10)-e5 50 41 2.5 0.16 0.67 529804 k-d(10)-kekee 50 40 2.3 0.14 0.62 534796 elck-d(10)-kke 50 37 2.6 0.15 0.51 540162 eek-d(10)-kke 50 42 2.4 0.15 0.60 540175 eek-d(10)-kke 50 39 2.2 0.11 0.62 540182 eek-d(10)-kke 50 41 2.6 0.16 0.61 540191 eek-d(10)-kke 50 40 2.4 0.13 0.60 e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

Example 35 Tolerability of Antisense Oligonucleotides Targeting Human Target-X in Sprague-Dawley Rats

Sprague-Dawley rats were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.

Treatment

Two groups of 4 male 7-8 week old Sprague-Dawley rats each were injected subcutaneously twice a week for 6 weeks with 50 mg/kg of ISIS 407935 and ISIS 490279. Another seven groups of 4 male 6-8 week old Sprague-Dawley rats each were injected subcutaneously twice a week for 6 weeks with 25 mg/kg of ISIS 473589, ISIS 529804, ISIS 534796, ISIS 540162, ISIS 540175, ISIS 540182, and ISIS 540191. One group of male Sprague-Dawley rats was injected subcutaneously twice a week for 6 weeks with PBS. The rats were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Plasma Chemistry Markers

To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in Table 41. Treatment with the all antisense oligonucleotides was tolerable in terms of plasma chemistry markers in this model.

TABLE 41 Effect of antisense oligonucleotide treatment on liver function in Sprague-Dawley rats Dose (mg/kg/ ALT AST BUN Bilirubin Motif wk) (IU/L) (IU/L) (mg/dL) (mg/dL) PBS — — 71 83 19 0.2 407935 e5-d(10)-e5 100 74 96 22 0.2 490279 kdkdk-d(9)-ee 100 96 181 22 0.4 473589 e5-d(10)-e5 50 57 73 21 0.2 529804 k-d(10)-kekee 50 54 78 21 0.2 534796 ekk-d(10)-kke 50 68 98 22 0.2 540162 eek-d(10)-kke 50 96 82, 21 0.1 540175 eek-d(10)-kke 50 55 73 18 0.2 540182 eek-d(10)-kke 50 45 87 21 0.2 540191 eek-d(10)-kke 50 77 104 21 0.2 e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

Body and Organ Weights

Body weights, as well as liver, heart, lungs, spleen and kidney weights were measured at the end of the study, and are presented in Table 42. Treatment with all the antisense oligonucleotides was tolerable in terms of body and organ weights in this model.

TABLE 42 Body and organ weights (grams) of Sprague-Dawley rats Dose (mg/kg/ Body Motif wk) weight Liver Spleen Kidney PBS — — 443 16 0.8 3.5 ISIS 407935 e5-d(10)-e5 100 337 14 1.8 3.2 ISIS 490279 kdkdk-d(9)-ee 100 365 18 2.2 2.9 ISIS 473589 e5-d(10)-e5 50 432 18 1.3 3.3 ISIS 529804 k-d(10)-kekee 50 429 18 2.2 3.4 ISIS 534796 ekk-d(10)-kke 50 434 15 1.4 3.3 ISIS 540162 eek-d(10)-kke 50 446 18 1.1 3.3 ISIS 540175 eek-d(10)-kke 50 467 16 1.0 3.5 ISIS 540182 eek-d(10)-kke 50 447 22 2.5 4.5 ISIS 540191 eek-d(10)-kke 50 471 21 1.4 3.9 e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

Example 36 Dose-Dependent Antisense Inhibition of Human Target-X in Cynomolgos Monkey Primary Hepatocytes

Antisense oligonucleotides selected from the studies described above were tested at various doses in cynomolgous monkey primary hepatocytes. Cells were plated at a density of 35,000 cells per well and transfected using electroporation with 0.009 μM, 0.03 μM, 0.08 μM, 0.25 μM, 0.74 μM, 2.22 μM, 6.67 μM, and 20.00 μM concentrations of antisense oligonucleotide, as specified in Table 43. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Target-X primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells. As illustrated in Table 43, Target-X mRNA levels were reduced in a dose-dependent manner with some of the antisense oligonucleotides that are cross-reactive with the rhesus monkey genomic sequence designated herein as Target-X.

TABLE 43 Dose-dependent antisense inhibition of Target-X in cynomolgous monkey primary hepatocytes using electroporation ISIS 0.009 0.03 0.08 0.25 0.74 2.22 6.67 20.00 No μM μM μM μM μM μM μM μM 407935 10 18 15 29 56 73 82 88 490279 19 12 13 0 6 18 27 22 473589 5 10 19 42 64 76 88 92 529804 10 3 23 25 57 80 86 91 534796 0 28 23 49 71 81 87 90 540162 9 14 9 6 13 13 11 31 540175 0 4 12 9 10 16 12 22 540182 0 7 0 6 36 12 10 0 540191 6 7 0 0 0 0 21 42

Example 37 Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B Cells

Antisense oligonucleotides from the study described above were also tested at various doses in Hep3B cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.009 μM, 0.03 μM, 0.08 μM, 0.25 μM, 0.74 μM, 2.22 μM, 6.67 μM, and 20.00 μM concentrations of antisense oligonucleotide, as specified in Table 44. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Target-X primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells. As illustrated in Table 44, Target-X mRNA levels were reduced in a dose-dependent manner with several of the antisense oligonucleotides.

TABLE 44 Dose-dependent antisense inhibition of Target-X in Hep3B cells using electroporation ISIS 0.009 0.03 0.08 0.25 0.74 2.22 6.67 20.00 IC₅₀ No μM μM μM μM μM μM μM μM (μM) 407935 3 9 11 35 64 83 87 93 4.5 473244 20 33 50 69 77 89 7 14 0.9 473589 0 14 23 44 74 88 90 94 2.7 490279 0 5 7 15 25 61 76 78 11.6 515533 0 12 21 36 63 78 88 94 3.6 515952 0 12 27 57 76 89 93 94 2.2 516066 6 0 12 26 52 70 81 86 6.0 529459 0 4 24 40 61 78 88 94 3.5 529553 9 7 17 40 58 74 87 93 4.6 529804 0 3 3/1 64 83 89 93 95 2.0 534796 8 18 43 67 82 89 95 96 1.4 537806 6 11 5 20 37 69 79 86 7.1 540162 18 33 63 75 87 91 91 92 0.7 540175 10 25 55 76 86 89 89 93 1.0 540182 13 36 61 75 84 88 90 93 0.7 540191 3 12 28 61 79 80 88 94 2.2

Example 38 Efficacy of Antisense Oligonucleotides Targeting Human Target-X in Transgenic Mice

Transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for efficacy.

Treatment

Eight groups of 3 transgenic mice each were injected subcutaneously twice a week for 3 weeks with 20 mg/kg/week, 10 mg/kg/week, 5 mg/kg/week, or 2.5 mg/kg/week of ISIS 407935or ISIS 490279. Another 24 groups of 3 transgenic mice each were subcutaneously twice a week for 3 weeks with 5 mg/kg/week, 2.5 mg/kg/week, 1.25 mg/kg/week, or 0.625 mg/kg/week of ISIS 473589, ISIS 529804, ISIS 534796, ISIS 540162, ISIS 540175, or ISIS 540191. One group of mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

RNA Analysis

RNA was extracted from plasma for real-time PCR analysis of Target-X, using primer probe set RTS2927. The mRNA levels were normalized using RIBOGREEN®. As shown in Table 45, several antisense oligonucleotides achieved reduction of human Target-X over the PBS control. Results are presented as percent inhibition of Target-X, relative to control. Treatment with newly designed 2′-MOE gapmer, ISIS 490279, caused greater reduction in human Target-X mRNA levels than treatment with ISIS 407935, the 2′-MOE gapmer from the earlier publication. Treatment with several of the newly designed oligonucleotides also caused greater reduction in human Target-X mRNA levels than treatment with ISIS 407935.

TABLE 45 Percent inhibition of Target-X mRNA in transgenic mice Dose % ISIS No Motif (mg/kg/wk) inhibition 407935 e5-d(10)-e5 20.0 85 10.0 57 5.0 45 2.5 28 490279 kdkdk-d(9)-ee 20.0 88 10.0 70 5.0 51 2.5 33 473589 e5-d(10)-e5 5.00 80 2.50 62 1.25 44 0.625 25 529804 k-d(10)-kekee 5.00 55 2.50 41 1.25 0 0.625 1 534796 ekk-d(10)-kke 5.00 56 2.50 41 1.25 5 0.625 0 540162 eek-d(10)-kke 5.00 97 2.50 92 1.25 69 0.625 78 540175 eek-d(10)-kke 5.00 95 2.50 85 1.25 65 0.625 55 540182 eek-d(10)-kke 5.00 97 2.50 83 1.25 54 0.625 10 540191 eek-d(10)-kke 5.00 91 2.50 74 1.25 58 0.625 34 e = 2'-MOE, k = cEt, d = 2'-deoxynucleoside

Protein Analysis

Plasma protein levels of Target-X were estimated using a Target-X ELISA kit (purchased from Hyphen Bio-Med). As shown in Table 46, several antisense oligonucleotides achieved reduction of human Target-X over the PBS control. Results are presented as percent inhibition of Target-X, relative to control.

TABLE 46 Percent inhibition of Target-X plasm protein levels in transgenic mice Dose % ISIS No Motif (mg/kg/wk) inhibition 407935 e5-d(10)-e5 20 65 10 47 5 0 2.5 3 490279 kdkdk-d(9)-ee 20 91 10 75 5 31 2.5 23 473589 e5-d(10)-e5 5 78 2.5 40 1.25 6 0.625 0 529804 k-d(10)-kekee 5 50 2.5 36 1.25 0 0.625 8 534796 ekk-d(10)-kke 5 45 2.5 26 1.25 0 0.625 8 540162 eek-d(10)-kke 5 98 2.5 96 1.25 78 0.625 74 540175 eek-d(10)-kke 5 93 2.5 83 1.25 49 0.625 24 540182 eek-d(10)-kke 5 97 2.5 71 1.25 50 0.625 0 540191 eek-d(10)-kke 5 97 2.5 74 1.25 46 0.625 25 e = 2'-MOE, k = cEt, d = 2'-deoxynucleoside

Example 39 Effect of ISIS Antisense Oligonucleotides Targeting Human Target-X in Cynomolgus Monkeys

Cynomolgus monkeys were treated with ISIS antisense oligonucleotides selected from studies described above, including ISIS 407935, ISIS 490279, ISIS 473589, ISIS 529804, ISIS 534796, ISIS 540162, ISIS 540175, ISIS 540182, and ISIS 540191. Antisense oligonucleotide efficacy was evaluated. ISIS 407935, from the earlier publication, was included in the study for comparison.

Treatment

Prior to the study, the monkeys were kept in quarantine for at least a 30-day period, during which the animals were observed daily for general health. Standard panels of serum chemistry and hematology, examination of fecal samples for ova and parasites, and a tuberculosis test were conducted immediately after the animals' arrival to the quarantine area. The monkeys were 2-4 years old at the start of treatment and weighed between 2 and 4 kg. Ten groups of four randomly assigned male cynomolgus monkeys each were injected subcutaneously with ISIS oligonucleotide or PBS using a stainless steel dosing needle and syringe of appropriate size into one of 4 sites on the back of the monkeys; each site used in clock-wise rotation per dose administered. Nine groups of monkeys were dosed four times a week for the first week (days 1, 3, 5, and 7) as loading doses, and subsequently once a week for weeks 2-12, with 35 mg/kg of ISIS 407935, ISIS 490279, ISIS 473589, ISIS 529804, ISIS 534796, ISIS 540162, ISIS 540175, ISIS 540182, or ISIS 540191. A control group of cynomolgus monkeys was injected with PBS subcutaneously thrice four times a week for the first week (days 1, 3, 5, and 7), and subsequently once a week for weeks 2-12. The protocols described in the Example were approved by the Institutional Animal Care and Use Committee (IACUC).

Hepatic Target Reduction RNA Analysis

On day 86, RNA was extracted from liver tissue for real-time PCR analysis of Target-X using primer probe set RTS2927. Results are presented as percent inhibition of Target-X mRNA, relative to PBS control, normalized to RIBOGREEN® or to the house keeping gene, GAPDH. As shown in Table 52, treatment with ISIS antisense oligonucleotides resulted in reduction of Target-X mRNA in comparison to the PBS control.

TABLE 52 Percent Inhibition of cynomolgous monkey Target-X mRNA in the cynomolgus monkey liver relative to the PBS control RTS2927/ RTS2927/ ISIS No Motif Ribogreen GAPDH 407935 e5-d(10)-e5 90 90 490279 kdkdk-d(9)-ee 72 66 473589 e5-d(10)-e5 96 96 529804 k-d(10)-kekee 90 87 534796 ekk-d(10)-kke 80 78 540162 eek-d(10)-kke 66 58 540175 eek-d(10)-kke 68 66 540182 eek-d(10)-kke 0 0 540191 eek-d(10)-kke 34 14 e = 2'-MOE, k = cEt, d = 2-deoxynucleoside

Protein Levels and Activity Analysis

Plasma Target-X levels were measured prior to dosing, and on day 3, day 5, day 7, day 16, day 30, day 44, day 65, and day 86 of treatment. Target-X activity was measured using Target-X deficiuent plasma. Approximately 1.5 mL of blood was collected from all available study animals into tubes containing 3.2% sodium citrate. The samples were placed on ice immediately after collection. Collected blood samples were processed to platelet poor plasma and the tubes were centrifuged at 3,000 rpm for 10 min at 4° C. to obtain plasma.

Protein levels of Target-X were measured by a Target-X elisa kit (purchased from Hyphen BioMed). The results are presented in Table 53.

TABLE 53 Plasma Target-X protein levels (% reduction compared to the baseline) in the cynomolgus monkey plasma ISIS Day Day Day Day Day Day Day Day No 3 5 7 16 30 44 65 86 407935 21 62 69 82 84 85 84 90 490279 0 29 35 30 38 45 51 58 473589 12 67 85 97 98 98 98 98 529804 19 65 76 87 88 89 90 90 534796 1 46 54 64 64 67 66 70 540162 0 24 26 37 45 49 49 50 540175 0 28 36 38 47 52 55 55 540182 0 17 8 0 0 0 5 0 540191 0 12 4 0 0 4 9 10

Example 40 Inhibition of Chimeric Antisense Oligonucleotides Targeting Target-Y

A series of modified oligonucleotides were designed based on the parent gapmer, ISIS XXXX01, wherein the central gap region contains ten 2′-deoxyribonucleosides. These modified oligonucleotides were designed by having the central gap region shortened to nine, eight or seven 2′-deoxynucleosides and by introducing 2′-O-methoxyethyl (MOE) modifications at one or both wing regions. The newly designed oligonucleotides (except for ISIS XXXX09) were evaluated for their effects in reducing Target-Y mRNA levels in vitro.

The gapmers and their motifs are described in Table 52. The internucleoside linkages throughout each gapmer are phosphorothioate linkages (P═S). Nucleosides followed by a subscript “d” indicate 2′-deoxynucleosides. Nucleosides followed by a subscript “e” indicate 2′-O-methoxyethyl (MOE) nucleosides.

Nucleosides followed by a subscript “k” indicate constrained ethyl (cEt) nucleosides. “N” indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).

The newly designed gapmers were tested in vitro. Mouse primary hepatocytes were plated at a density of 20,000 cells per well and transfected using electroporation with 15 μM concentration of antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-Y mRNA levels were measured by quantitative real-time PCR. Mouse Target-Y primer probe set RTS2898 was used to measure mRNA levels. Target-Y mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. The results in Table 53 are presented as Target-Y mRNA expression relative to untreated control cells (% UTC).

The parent gapmer, ISIS XXXX01 was included in the study as a bench mark oligonucleotide against which the activity of the newly designed gapmers targeting Target-Y could be compared.

As illustrated, most of the newly designed gapmers showed similar activity as compared to ISIS 464917.

TABLE 52 Chimeric antisense oligonucleotides targeting Target-Y ISIS Gap Wing chemistry SEQ ID NO. Sequence (5′ to 3′) Motif chemistry 5′ 3′ NO XXXX01 N_(k)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d) 3-10-3 Full deoxy kkk kkk 19 N_(d)N_(d)N_(k)N_(k)N_(k) XXXX02 N_(k)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d) 3-10-3 Full deoxy kkk eee 19 N_(d)N_(d)N_(e)N_(e)N_(e) XXXX03 N_(e)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d) 3-10-3 Full deoxy ekk kke 19 N_(d)N_(d)N_(k)N_(k)N_(e) XXXX04 N_(e)N_(e)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d) 4-9-3 Full deoxy eekk kke 19 N_(d)N_(d)N_(k)N_(k)N_(e) XXXX05 N_(e)N_(e)N_(e)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d) 5-8-3 Full deoxy eeekk kke 19 N_(d)N_(d)N_(k)N_(k)N_(e) XXXX06 N_(e)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d) 3-9-4 Full deoxy ekk kkee 19 N_(d)N_(k)N_(k)N_(e)N_(e) XXXX07 N_(e)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d) 3-8-5 Full deoxy ekk kkeee 19 N_(k)N_(k)N_(e)N_(e)N_(e) XXXX08 N_(e)N_(e)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d) 4-8-4 Full deoxy eekk kkee 19 N_(d)N_(k)N_(k)N_(e)N_(e) XXXX09 N_(e)N_(e)N_(e)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d) 5-7-4 Full deoxy eeekk kkee 19 N_(d)N_(k)N_(k)N_(e)N_(e) XXXX10 N_(e)N_(e)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d) 4-7-5 Full deoxy eekk kkeee 19 N_(k)N_(k)N_(e)N_(e)N_(e) e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

TABLE 53 Inhibition of modified oligonucleotides targeting Target-Y % UTC Gap Wing chemistry ISIS NO 15 μM Motif chemistry 5' 3' XXXX01 8.5 3-10-3 Full deoxy kkk kkk XXXX02 9.1 3-10-3 Full deoxy kkk eee XXXX03 8.3 3-10-3 Full deoxy ekk kke XXXX04 7.1 4-9-3 Full deoxy eekk kke XXXX05 8.6 5-8-3 Full deoxy eeekk kke XXXX06 7.4 3-9-4 Full deoxy ekk kkee XXXX07 8.5 3-8-5 Full deoxy ekk kkeee XXXX08 12.5 4-8-4 Full deoxy eekk kkee XXXX10 11.2 4-7-5 Full deoxy eekk kkeee e = 2'-MOE, k = cEt, d = 2'-deoxynucleoside

Example 41 Dose-Dependent Inhibition of Chimeric Antisense Oligonucleotides Targeting Target-Y

Additional chimeric antisense oligonucleotides were designed based on the parent gapmer, ISIS XXXX11, wherein the central gap region contains ten 2′-deoxynucleosides. These modified oligonucleotides were designed in a similar manner as the chimeric antisense oligonucleotides described in Example 40 and were evaluated for their effect in reducing Target-Y mRNA levels in vitro.

The gapmers and their motifs are described in Table 54. The internucleoside linkages throughout each gapmer are phosphorothioate linkages (P═S). Nucleosides followed by a subscript “d” indicate 2′-deoxynucleosides. Nucleosides followed by a subscript “e” indicate 2′-O-methoxyethyl (MOE) nucleosides. Nucleosides followed by a subscript “k” indicate constrained ethyl (cEt) nucleosides. “N” indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).

The newly designed gapmers were tested in vitro. Mouse primary hepatocytes were plated at a density of 20,000 cells per well and transfected using electroporation with 0.6 μM, 3.0 μM and 15 μM concentrations of antisense oligonucleotides. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-Y mRNA levels were measured by quantitative real-time PCR. Mouse Target-Y primer probe set RTS2898 was used to measure mRNA levels. Target-Y mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. The results in Table 55 are presented as Target-Y mRNA expression relative to untreated control cells (% UTC).

The parent gapmer, ISIS XXXX11 was included in the study as a bench mark oligonucleotide against which the activity of the newly designed gapmers targeting Target-Y could be compared.

As illustrated in Table 55, several of the newly designed gapmers exhibited similar activity as compared to ISIS XXXX11. The data also confirms that Target-Y mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 54 Chimeric antisense oligonucleotides targeting Target-Y ISIS Gap Wing chemistry SEQ ID NO. Sequence (5′ to 3′) Motif chemistry 5′ 3′ NO. XXXX11 N_(k)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d) 3-10-3 Full deoxy kkk kkk 19 N_(d)N_(k)N_(k)N_(k) XXXX12 N_(k)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d) 3-10-3 Full deoxy kkk eee 19 N_(d)N_(e)N_(e)N_(e) XXXX13 N_(e)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d) 3-10-3 Full deoxy ekk kke 19 N_(d)N_(k)N_(k)N_(e) XXXX14 N_(e)N_(e)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d) 4-9-3 Full deoxy eekk kke 19 N_(d)N_(k)N_(k)N_(e) XXXX15 N_(e)N_(e)N_(e)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d) 5-8-3 Full deoxy eeekk kke 19 N_(d)N_(k)N_(k)N_(e) XXXX16 N_(e)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d) 3-9-4 Full deoxy ekk kkee 19 N_(k)N_(k)N_(e)N_(e) XXXX17 N_(e)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(k) 3-8-5 Full deoxy ekk kkeee 19 N_(k)N_(e)N_(e)N_(e) XXXX18 N_(e)N_(e)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d) 4-8-4 Full deoxy eekk kkee 19 N_(k)N_(k)N_(e)N_(e) XXXX19 N_(e)N_(e)N_(e)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d) 5-7-4 Full deoxy eeekk kkee 19 N_(k)N_(k)N_(e)N_(e) XXXX20 N_(e)N_(e)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(k) 4-7-5 Full deoxy eekk kkeee 19 N_(k)N_(e)N_(e)N_(e) e = 2′-MOE k = cEt, d = 2′-deoxynucleoside

TABLE 55 Dose-dependent inhibition of chimeric antisense oligonucleotides targeting Target-Y % UTC Wing 0.6 3.0 15 Gap chemistry ISIS NO μM μM μM Motif chemistry 5′ 3′ XXXX11 19.4 14.1 12.5 3-10-3 Full deoxy kkk kkk XXXX12 23.4 12.5 9.9 3-10-3 Full deoxy kkk eee XXXX13 29.8 13.7 11.2 3-10-3 Full deoxy ekk kke XXXX14 28.3 15.5 11.6 4-9-3 Full deoxy eekk kke XXXX15 41.3 16.7 11.6 5-8-3 Full deoxy eeekk kke XXXX16 31.6 16.7 11.7 3-9-4 Full deoxy ekk kkee XXXX17 39.2 16.8 11.1 3-8-5 Full deoxy ekk kkeee XXXX18 40.5 18.2 13.6 4-8-4 Full deoxy eekk kkee XXXX19 118.4 123.8 13.3 5-7-4 Full deoxy eeekk kkee XXXX20 52.3 27.6 12.4 4-7-5 Full deoxy eekk kkeee Saline = 100 e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

Example 42 Dose-Dependent Inhibition of Chimeric Antisense Oligonucleotides Targeting Target-Y

Additional chimeric oligonucleotides were designed based on the parent gapmer, ISIS XXXX01, wherein the central gap region contains ten 2′-deoxynucleosides. These modified oligonucleotides were designed by having the central gap region shortened to eight 2′-deoxynucleosides and by introducing one or more 2′-O-methoxyethyl (MOE) modification(s) at the 3′ wing region. The modified oligonucleotides designed by microwalk were evaluated for their effects in reducing Target-Y mRNA levels in vitro.

The gapmers and their motifs are described in Table 56. The internucleoside linkages throughout each gapmer are phosphorothioate linkages (P═S). Nucleosides followed by a subscript “d” indicate 2′-deoxynucleoside. Nucleosides followed by a subscript “e” indicate 2′-O-methoxyethyl (MOE) nucleosides. Nucleosides followed by a subscript “k” indicate constrained ethyl (cEt) nucleosides. “N” indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).

The newly designed gapmers were tested in vitro. Mouse primary hepatocytes were plated at a density of 20,000 cells per well and transfected using electroporation with 0.6 μM, 3.0 μM and 15 μM concentrations of antisense oligonucleotides. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-Y mRNA levels were measured by quantitative real-time PCR. Mouse Target-Y primer probe set RTS2898 was used to measure mRNA levels. Target-Y mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. The results in Table 57 are presented as Target-Y mRNA expression relative to untreated control cells (% UTC).

The parent gapmer, ISIS XXXX01 was included in the study as a bench mark oligonucleotide against which the activity of the newly designed gapmers targeting Target-Y could be compared.

As illustrated in Table 57, most of the newly designed gapmers demonstrated improvement in activity at low concentrations (0.6 μM and 3.0 μM) as compared to ISIS XXXX01. The data also confirms that Target-Y mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 56 Chimeric antisense oligonucleotides designed by microwalk targeting Target-Y ISIS Gap Wing chemistry SEQ ID NO. Sequence (5′ to 3′) Motif chemistry 5′ 3′ NO. XXXX01 N_(k)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d) 3-10-3 Full deoxy kkk kkk 19 N_(d)N_(k)NkN_(k) XXXX21 N_(k)NkN_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d) 3-10-3 Full deoxy kkk eee 19 N_(d)N_(e)N_(e)N_(e) XXXX22 N_(k)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(k) 3-8-5 Full deoxy kkk keeee 19 N_(e)N_(e)N_(e)N_(e) XXXX23 N_(k)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(k) 3-8-5 Full deoxy kkk keeee 19 N_(e)N_(e)N_(e)N_(e) XXXX24 N_(k)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(k) 3-8-5 Full deoxy kkk keeee 19 N_(e)N_(e)N_(e)N_(e) XXXX25 N_(k)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(k) 3-8-5 Full deoxy kkk keeee 19 N_(e)N_(e)N_(e)N_(e) XXXX26 N_(k)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(k) 3-8-5 Full deoxy kkk keeee 19 N_(e)N_(e)N_(e)N_(e) XXXX27 N_(k)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(k) 3-8-5 Full deoxy kkk keeee 19 N_(e)N_(e)N_(e)N_(e) XXXX28 N_(k)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(k) 3-8-5 Full deoxy kkk keeee 19 N_(e)N_(e)N_(e)N_(e) XXXX29 N_(k)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(k) 3-8-5 Full deoxy kkk keeee 19 N_(e)N_(e)N_(e)N_(e) XXXX30 N_(k)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(k) 3-8-5 Full deoxy kkk keeee 19 N_(e)N_(e)N_(e)N_(e) e = 2′-MOE k = cEt, d = 2′-deoxynucleoside

TABLE 57 Dose-dependent inhibition of chimeric antisense oligonucleotides designed by microwalk targeting Target-Y % UTC Wing 0.6 3.0 15 Gap chemistry ISIS NO μM μM μM Motif chemistry 5′ 3′ XXXX01 83.9 94.3 8.5 3-10-3 Full deoxy kkk kkk XXXX21 39.8 21.2 9.1 3-10-3 Full deoxy kkk eee XXXX22 52.5 35.1 13.0 3-8-5 Full deoxy kkk keeee XXXX23 60.7 40.9 13.6 3-8-5 Full deoxy kkk keeee XXXX24 52.3 23.8 7.3 3-8-5 Full deoxy kkk keeee XXXX25 58.9 32.1 9.3 3-8-5 Full deoxy kkk keeee XXXX26 41.7 21.1 8.8 3-8-5 Full deoxy kkk keeee XXXX27 45.6 25.2 8.5 3-8-5 Full deoxy kkk keeee XXXX28 39.1 20.1 9.2 3-8-5 Full deoxy kkk keeee XXXX29 61.4 28.4 9.9 3-8-5 Full deoxy kkk keeee XXXX30 81.3 52.2 16.2 3-8-5 Full deoxy kkk keeee Saline = 100 e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

Example 43 Dose-Dependent Inhibition of Chimeric Antisense Oligonucleotides Targeting Target-Y

Additional chimeric oligonucleotides were designed based on the parent gapmer, ISIS XXXX11, wherein the central gap region contains ten 2′-deoxynucleosides. These modified oligonucleotides were designed by in the same manner as the oligonucleotides described in Example 42. The modified oligonucleotides designed by microwalk were evaluated for their effects in reducing Target-Y mRNA levels in vitro.

The gapmers and their motifs are described in Table 58. The internucleoside linkages throughout each gapmer are phosphorothioate linkages (P═S). Nucleosides followed by a subscript “d” indicate 2′-deoxynucleoside. Nucleosides followed by a subscript “e” indicate 2′-O-methoxyethyl (MOE) nucleosides. Nucleosides followed by a subscript “k” indicate constrained ethyl (cEt) nucleosides. “N” indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).

The newly designed gapmers were tested in vitro. Mouse primary hepatocytes were plated at a density of 20,000 cells per well and transfected using electroporation with 0.6 μM, 3.0 μM and 15 μM concentrations of antisense oligonucleotides. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-Y mRNA levels were measured by quantitative real-time PCR. Mouse Target-Y primer probe set RTS2898 was used to measure mRNA levels. Target-Y mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. The results in Table 59 are presented as Target-Y mRNA expression relative to untreated control cells (% UTC).

The parent gapmer, ISIS XXXX11 was included in the study as a bench mark oligonucleotide against which the activity of the newly designed gapmers targeting Target-Y could be compared.

TABLE 58 Chimeric antisense oligonucleotides designed by microwalk targeting Target-Y ISIS Gap Wing chemistry SEQ ID NO. Sequence (5′ to 3′) Motif chemistry 5′ 3′ NO. XXXX11 N_(k)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d) 3-10-3 Full deoxy kkk kkk 19 N_(d)N_(k)N_(k)N_(k) XXXX31 N_(k)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(k) 3-8-5 Full deoxy kkk keeee 19 N_(e)N_(e)N_(e)N_(e) XXXX32 N_(k)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(k) 3-8-5 Full deoxy kkk keeee 19 N_(e)N_(e)N_(e)N_(e) XXXX33 N_(k)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(k) 3-8-5 Full deoxy kkk keeee 19 N_(e)N_(e)N_(e)N_(e) XXXX34 N_(k)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(k) 3-8-5 Full deoxy kkk keeee 19 N_(e)N_(e)N_(e)N_(e) XXXX35 N_(k)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(k) 3-8-5 Full deoxy kkk keeee 19 N_(e)N_(e)N_(e)N_(e) XXXX36 N_(k)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(k) 3-8-5 Full deoxy kkk keeee 19 N_(e)N_(e)N_(e)N_(e) XXXX37 N_(k)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(k) 3-8-5 Full deoxy kkk keeee 19 N_(e)N_(e)N_(e)N_(e) XXXX38 N_(k)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(k) 3-8-5 Full deoxy kkk keeee 19 N_(e)N_(e)N_(e)N_(e) XXXX39 N_(k)N_(k)N_(k)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(d)N_(k) 3-8-5 Full deoxy kkk keeee 19 N_(e)N_(e)N_(e)N_(e) e = 2′-MOE k = cEt, d = 2′-deoxynucleoside

TABLE 59 Dose-dependent inhibition of chimeric antisense oligonucleotides designed by microwalk targeting Target-Y % UTC Wing 0.6 3.0 15 Gap chemistry ISIS NO μM μM μM Motif chemistry 5′ 3′ XXXX11 19.4 14.1 12.5 3-10-3 Full deoxy kkk kkk XXXX31 50.5 23.0 14.2 3-8-5 Full deoxy kkk keeee XXXX32 50.2 19.4 8.7 3-8-5 Full deoxy kkk keeee XXXX33 55.2 19.3 11.9 3-8-5 Full deoxy kkk keeee XXXX34 53.3 15.3 11.9 3-8-5 Full deoxy kkk keeee XXXX35 35.5 18.7 11.1 3-8-5 Full deoxy kkk keeee XXXX36 39.7 22.3 16.8 3-8-5 Full deoxy kkk keeee XXXX37 24.1 16.7 9.5 3-8-5 Full deoxy kkk keeee XXXX38 26.3 13.8 10.9 3-8-5 Full deoxy kkk keeee XXXX39 36.9 16.4 10.4 3-8-5 Full deoxy kkk keeee Saline = 100 e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

Example 44 Dose-Dependent Inhibition of Chimeric Antisense Oligonucleotides Targeting PTEN

A series of modified oligonucleotides were designed based on the parent gapmer, ISIS 482050, wherein the central gap region contains ten 2′-deoxynucleosides. These modified oligonucleotides were designed by having the central gap region shortened to nine, eight or seven 2′-deoxynucleosides and by introducing 2′-O-methoxyethyl (MOE) modifications at one or both wing regions. The newly designed oligonucleotides were evaluated for their effecst in reducing PTEN mRNA levels in vitro.

The gapmers and their motifs are described in Table 60. The internucleoside linkages throughout each gapmer are phosphorothioate linkages (P═S). Nucleosides followed by a subscript “d” indicate 2′-deoxynucleosides. Nucleosides followed by a subscript “e” indicate 2′-O-methoxyethyl (MOE) nucleosides. Nucleosides followed by a subscript “k” indicate constrained ethyl (cEt) nucleosides. “N” indicates modified or naturally occurring nucleohases (A, T, C, G, U, or 5-methyl C).

The newly designed gapmers were tested in vitro. Mouse primary hepatocytes were plated at a density of 20,000 cells per well and transfected using electroporation with 0.6 μM, 3.0 μM and 15 μM concentrations of antisense oligonucleotides. After a treatment period of approximately 24 hours, RNA was isolated from the cells and PTEN mRNA levels were measured by quantitative real-time PCR. Mouse PTEN primer probe set RTS186 was used to measure mRNA levels. PTEN mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. The results in Table 61 are presented as PTEN mRNA expression relative to untreated control cells (% UTC).

The parent gapmer, ISIS 482050 was included in the study as a bench mark oligonucleotide against which the activity of the newly designed gapmers targeting PTEN could be compared.

TABLE 60 Chimeric antisense oligonucleotides targeting PTEN ISIS Gap Wing chemistry SEQ ID NO. Sequence (5′ to 3′) Motif chemistry 5′ 3′ NO. 482050 A_(k)T_(k) ^(m)C_(k)A_(d)T_(d)G_(d)G_(d) ^(m)C_(d)T_(d)G_(d) ^(m)C_(d) 3-10-3 Full deoxy kkk kkk 23 A_(d)G_(d) ^(m)C_(k)T_(k)T_(k) 508033 A_(k)T_(k) ^(m)CkA_(d)T_(d)G_(d)G_(d) ^(m)C_(d)T_(d)G_(d) ^(m)C_(d) 3-10-3 Full deoxy kkk eee 23 A_(d)G_(d) ^(m)C_(e)T_(e)T_(e) 573351 A_(e)T_(k) ^(m)C_(k)A_(d)T_(d)G_(d)G_(d) ^(m)C_(d)T_(d)G_(d) ^(m)C_(d) 3-10-3 Full deoxy ekk kke 23 A_(d)G_(d) ^(m)C_(k)T_(k)T_(e) 573352 A_(e)T_(e) ^(m)C_(k)A_(k)T_(d)G_(d)G_(d) ^(m)C_(d)T_(d)G_(d) ^(m)C_(d) 4-9-3 Full deoxy eekk kke 23 A_(d)G_(d) ^(m)C_(k)T_(k)T_(e) 573353 A_(e)T_(e) ^(m)C_(e)A_(k)T_(k)G_(d)G_(d) ^(m)C_(d)T_(d)G_(d) ^(m)C_(d) 5-8-3 Full deoxy eeekk kke 23 A_(d)G_(d) ^(m)C_(k)T_(k)T_(e) 573355 A_(e)T_(k) ^(m)C_(k)A_(d)T_(d)G_(d)G_(d)C_(d)T_(d)G_(d) ^(m)C_(d) 3-9-4 Full deoxy ekk kkee 23 A_(d)G_(k) ^(m)C_(k)T_(e)T_(e) 573356 A_(e)T_(k) ^(m)C_(k)A_(d)T_(d)G_(d)G_(d) ^(m)C_(d)T_(d)G_(d) ^(m)C_(d) 3-8-5 Full deoxy ekk kkeee 23 A_(k)G_(k) ^(m)G_(k) ^(m)C_(e)T_(e)T_(e) 573357 A_(k)T_(k) ^(m)C_(k)A_(d)T_(d)G_(d)G_(d) ^(m)C_(d)T_(d)G_(d) ^(m)C_(k) 3-7-6 Full deoxy ekk kkeeee 23 A_(k)G_(e) ^(m)C_(e)T_(e)T_(e) 573358 A_(e)T_(e) ^(m)C_(k)A_(k)T_(d)G_(d)G_(d) ^(m)C_(d)T_(d)G_(d) ^(m)C_(d) 4-8-4 Full deoxy eekk kkee 23 A_(d)G_(k) ^(m)C_(k)T_(e)T_(e) 573359 A_(e)T_(e) ^(m)C_(e)A_(k)T_(k)G_(d)G_(d) ^(m)C_(d)T_(d)G_(d) ^(m)C_(d) 5-7-4 Full deoxy eeekk kkee 23 A_(d)G_(k) ^(m)C_(k)T_(e)T_(e) 573360 A_(e)T_(e) ^(m)C_(k)A_(k)T_(d)G_(d)G_(d) ^(m)C_(d)T_(d)G_(d) ^(m)C_(d) 4-7-5 Full deoxy eekk kkeee 23 A_(k)G_(k) ^(m)C_(e)T_(e)T_(e) e = 2′-MOE k = cEt, d = 2′-deoxynucleoside

TABLE 61 Dose-response effect of chimeric antisense oligonucleotides targeting PTEN % UTC Wing 0.6 3.0 15 Gap chemistry ISIS NO μM μM μM Motif chemistry 5′ 3′ 482050 45.4 23.8 8.4 3-10-3 Full deoxy kkk kkk 508033 52.2 28.8 7.6 3-10-3 Full deoxy kkk eee 573351 66.0 24.0 12.4 3-10-3 Full deoxy ekk kke 573352 69.0 38.1 12.5 4-9-3 Full deoxy eekk kke 573353 59.8 36.5 13.8 5-8-3 Full deoxy eeekk kke 573355 52.1 37.4 11.4 3-9-4 Full deoxy ekk kkee 573356 52.9 46.4 15.4 3-8-5 Full deoxy ekk kkeee 573357 82.4 81.8 52.5 3-7-6 Full deoxy ekk kkeeee 573358 67.4 46.7 14.5 4-8-4 Full deoxy eekk kkee 573359 70.5 49.8 31.6 5-7-4 Full deoxy eeekk kkee 573360 62.2 50.8 17.6 4-7-5 Full deoxy eekk kkeee Saline = 100 e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

Example 45 Dose-Dependent Inhibition of Chimeric Antisense Oligonucleotides Targeting PTEN

Additional chimeric oligonucleotides were designed based on the parent gapmer, ISIS 482050, wherein the central gap region contains ten 2′-deoxynucleosides. These modified oligonucleotides were designed by having the central gap region shortened to eight 2′-deoxynucleosides and by introducing one or more 2′-O-methoxyethyl (MOE) modification(s) at the 3′ wing region. The modified oligonucleotides designed by microwalk were evaluated for their effects in reducing PTEN mRNA levels in vitro.

The gapmers and their motifs are described in Table 62. The internucleoside linkages throughout each gapmer are phosphorothioate linkages (P═S). Nucleosides followed by a subscript “d” indicate 2′-deoxynucleoside. Nucleosides followed by a subscript “e” indicate 2′-O-methoxyethyl (MOE) nucleosides. Nucleosides followed by a subscript “k” indicate constrained ethyl (cEt) nucleosides. ^(m)C indicates a 5-methyl nucleoside.

The newly designed gapmers were tested in vitro. Mouse primary hepatocytes were plated at a density of 20,000 cells per well and transfected using electroporation with 0.6 μM, 3.0 μM and 15 μM concentrations of antisense oligonucleotides. After a treatment period of approximately 24 hours, RNA was isolated from the cells and PTEN mRNA levels were measured by quantitative real-time PCR. Mouse PTEN primer probe set RTS186 was used to measure mRNA levels. PTEN mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. The results in Table 63 are presented as PTEN mRNA expression relative to untreated control cells (% UTC).

The parent gapmer, ISIS 482050 was included in the study as a bench mark oligonucleotide against which the activity of the newly designed gapmers targeting PTEN could be compared.

TABLE 62 Chimeric antisense oligonucleotides designed by microwalk targeting PTEN ISIS Gap Wing chemistry SEQ ID NO. Sequence (5′ to 3′) Motif chemistry 5′ 3′ NO. 482050 A_(k)T_(k) ^(m)C_(k)A_(d)T_(d)G_(d)G_(d) ^(m)C_(d)T_(d)G_(d) ^(m)C_(d) 3-10-3 Full deoxy kkk ldck 24 A_(d)G_(d) ^(m)C_(k)T_(k)T_(k) 573797 T_(k)G_(k)G_(k) ^(m)C_(d)T_(d)G_(d) ^(m)C_(d)A_(d)G_(d) ^(m)C_(d)T_(d) 3-8-5 Full deoxy kkk keeee 25 T_(k) ^(m)C_(e) ^(m)C_(e)G_(e)A_(e) 573798 A_(k)T_(k)G_(k)G_(d) ^(m)C_(d)T_(d)G_(d) ^(m)C_(d)A_(d)G_(d) ^(m)C_(d) 3-8-5 Full deoxy kkk keeee 26 T_(k)T_(e) ^(m)C_(e) ^(m)C_(e)G_(e) 573799 ^(m)C_(k)A_(k)T_(k)G_(d)G_(d) ^(m)C_(d)T_(d)G_(d) ^(m)C_(d)A_(d)G_(d) ^(m) 3-8-5 Full deoxy kkk keeee 27 C_(k)T_(e)T_(e) ^(m)C_(e) ^(m)C_(e) 573800 T_(k) ^(m)C_(k)A_(k)T_(d)G_(d)G_(d) ^(m)C_(d)T_(d)G_(d) ^(m)C_(d)A_(d) 3-8-5 Full deoxy kkk keeee 28 G_(k) ^(m)C_(e)T_(e)T_(e) ^(m)C_(e) 573801 A_(k)T_(k) ^(m)C_(k)A_(d)T_(d)G_(d)G_(d) ^(m)C_(d)T_(d)G_(d) ^(m)C_(d) 3-8-5 Full deoxy kkk ?+0 keeee 24 A_(k)G_(e) ^(m)C_(e)T_(e)T_(e) 573802 ^(m)C_(k)A_(k)T_(k) ^(m)C_(d)A_(d)T_(d)G_(d)G_(d) ^(m)C_(d)T_(d)G_(d) ^(m) 3-8-5 Full deoxy kkk keeee 29 C_(k)A_(e)G_(e) ^(m)C_(e)T_(e) 573803 ^(m)C_(k) ^(m)C_(k)A_(k)T_(d) ^(m)C_(d)A_(d)T_(d)G_(d)G_(d) ^(m)C_(d) 3-8-5 Full deoxy kkk keeee 30 T_(d)G_(k) ^(m)C_(e)A_(e)G_(e) ^(m)C_(e) 573804 T_(k) ^(m)C_(k) ^(m)C_(k)A_(d)T_(d) ^(m)C_(d)A_(d)T_(d)G_(d)G_(d) ^(m) 3-8-5 Full deoxy kkk keeee 31 C_(d)T_(k)G_(e) ^(m)C_(e)A_(e)G_(e) 573805 T_(k)T_(k) ^(m)C_(k) ^(m)C_(d)A_(d)T_(d) ^(m)C_(d)A_(d)T_(d)G_(d)G_(d) ^(m) 3-8-5 Full deoxy kkk keeee 32 C_(k)T_(e)G_(e) ^(m)C_(e)A_(e) e = 2′-MOE k = cEt, d = 2′-deoxynucleoside

TABLE 63 Dose-dependent inhibition of chimeric antisense oligonucleotides designed by microwalk targeting PTEN % UTC Wing 0.6 3.0 15 Gap chemistry ISIS NO μM μM μM Motif chemistry 5′ 3′ 482050 45.4 23.8 8.4 3-10-3 Full deoxy kkk kkk 573797 56.8 55.4 13.1 3-8-5 Full deoxy kkk keeee 573798 50.9 33.5 9.6 3-8-5 Full deoxy kkk keeee 573799 62.6 27.7 10.3 3-8-5 Full deoxy kkk keeee 573800 68.6 38.9 12.0 3-8-5 Full deoxy kkk keeee 573801 54.6 46.3 11.8 3-8-5 Full deoxy kkk keeee 573802 60.7 40.4 13.0 3-8-5 Full deoxy kkk keeee 573803 47.0 29.8 8.5 3-8-5 Full deoxy kkk keeee 573804 62.5 34.1 11.3 3-8-5 Full deoxy kkk keeee 573805 70.3 31.6 15.2 3-8-5 Full deoxy kkk keeee Saline = 100 e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

Example 46 Antisense Inhibition of Target-Z mRNA in HepG2 Cells

Antisense oligonucleotides were designed targeting a Target-Z nucleic acid and were tested for their effects on Target-Z mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. ISIS 146786, 509934, ISIS 509959, and ISIS 510100, were also included in these studies for comparison. Cultured HepG2 cells at a density of 28,000 cells per well were transfected using LipofectAMINE2000® with 70 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-Z mRNA levels were measured by quantitative real-time PCR. Viral primer probe set RTS3370 (forward sequence CTTGGTCATGGGCCATCAG, designated herein as SEQ ID NO: 33; reverse sequence CGGCTAGGAGTTCCGCAGTA, designated herein as SEQ ID NO: 34; probe sequence TGCGTGGAACCTTTTCGGCTCC, designated herein as SEQ ID NO: 35) was used to measure mRNA levels. Levels were also measured using primer probe set RTS3371 (forward sequence CCAAACCTTCGGACGGAAA, designated herein as SEQ ID NO: 36; reverse sequence TGAGGCCCACTCCCATAGG, designated herein as SEQ ID NO: 37; probe sequence CCCATCATCCTGGGCTTTCGGAAAAT, designated herein as SEQ ID NO: 38). Target-Z mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-Z, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides and their motifs are described in Tables 64-69. The gapmers are 16 nucleotides in length, wherein the central gap region comprises ten 2′-deoxynucleosides. Nucleosides followed by ‘k’ indicate constrained ethyl (cEt) nucleosides. Nucleosides followed by “e” indicate 2′-O-methoxyethyl (2′-MOE) nucleosides. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.

Each gapmer listed in Tables 64-69 is targeted to the viral genomic sequence, designated herein as Target-Z. The activity of the newly designed oligonucleotides was compared with ISIS 146786, ISIS 509934, ISIS 509959, and ISIS 510100.

TABLE 64 Inhibition of viral Target-Z mRNA levels by chimeric antisense oligonucleotidesmeasured with RTS3370 % ISIS No Motif inhibition 509934 eeeee-d(10)-eeeee 30 552787 ekk-d(10)-kke 57 552788 ekk-d(10)-kke 60 552789 ekk-d(10)-kke 67 552790 ekk-d(10)-kke 67 552791 ekk-d(10)-kke 65 552792 ekk-d(10)-kke 44 552793 ekkd(10)kke 0 552794 ekk-d(10)-kke 54 552795 ekk-d(10)-kke 55 552796 ekk-d(10)-kke 62 552797 ekk-d(10)-kke 59 552798 ekk-d(10)-kke 59 552799 ekk-d(10)-kke 58 552800 ekk-d(10)-kke 62 552801 ekk-d(10)-kke 65 552802 ekk-d(10)-kke 53 552803 ekk-d(10)-kke 67 552804 ekk-d(10)-kke 75 552805 ekk-d(10)-kke 72 552806 ekk-d(10)-kke 64 552807 ekk-d(10)-kke 68 552808 ekk-d(10)-kke 65 552809 ekk-d(10)-kke 60 552810 ekk-d(10)-kke 59 552811 ekk-d(10)-kke 64 552812 ekk-d(10)-kke 69 552813 ekk-d(10)-kke 64 552814 ekk-d(10)-kke 62 552815 ekk-d(10)-kke 61 552816 ekk-d(10)-kke 63 552817 ekk-d(10)-kke 42 552818 ekk-d(10)-kke 44 552819 ekk-d(10)-kke 56 552820 ekk-d(10)-kke 59 552821 ekk-d(10)-kke 76 552822 ekk-d(10)-kke 77 552823 ekk-d(10)-kke 73 552824 ekk-d(10)-kke 73 552825 ekk-d(10)-kke 51 552826 ekk-d(10)-kke 55 552827 ekk-d(10)-kke 67 552828 ekk-d(10)-kke 78 552829 ekk-d(10)-kke 72 552830 ekk-d(10)-kke 71 552831 ekk-d(10)-kke 69 552832 ekk-d(10)-kke 67 552833 ekk-d(10)-kke 65 552834 ekk-d(10)-kke 78 552835 ekk-d(10)-kke 70 552836 ekk-d(10)-kke 64 552837 ekk-d(10)-kke 65 552838 ekk-d(10)-kke 64 552839 ekk-d(10)-kke 60 552840 ekk-d(10)-kke 35 552841 ekk-d(10)-kke 62 552842 ekk-d(10)-kke 67 552843 ekk-d(10)-kke 77 552844 ekk-d(10)-kke 81 552845 ekk-d(10)-kke 63 552846 ekk-d(10)-kke 79 552847 ekk-d(10)-kke 47 552848 ekk-d(10)-kke 69 552849 ekk-d(10)-kke 59 552850 ekk-d(10)-kke 83 552851 ekk-d(10)-kke 90 552852 ekk-d(10)-kke 89 552853 ekk-d(10)-kke 83 552854 ekk-d(10)-kke 80 552855 ekk-d(10)-kke 75 552856 ekk-d(10)-kke 69 552857 ekk-d(10)-kke 68 552858 ekk-d(10)-kke 79 552859 ekk-d(10)-kke 79 552860 ekk-d(10)-kke 71 552861 ekk-d(10)-kke 68 552862 ekk-d(10)-kke 65 552863 ekk-d(10)-kke 70 552864 ekk-d(10)-kke 71 e = 2'-MOE, k = cEt, d = 2'-deoxynucleoside

TABLE 65 Inhibition of viral Target-Z mRNA levels by chimeric antisense oligonucleotidesmeasured with RTS3371 % ISIS No Motif inhibition 552787 ekk-d(10)-kke 53 552788 ekk-d(10)-kke 45 552789 ekk-d(10)-kke 75 552790 ekk-d(10)-kke 68 552791 ekk-d(10)-kke 51 552792 ekk-d(10)-kke 38 552793 ekk-d(10)-kke 0 552794 ekk-d(10)-kke 44 552795 ekk-d(10)-kke 56 552796 ekk-d(10)-kke 45 552797 ekk-d(10)-kke 46 552798 ekk-d(10)-kke 53 552799 ekk-d(10)-kke 48 552800 ekk-d(10)-kke 54 552801 ekk-d(10)-kke 63 552802 ekk-d(10)-kke 49 552803 ekk-d(10)-kke 71 552804 ekk-d(10)-kke 64 552805 ekk-d(10)-kke 70 552806 ekk-d(10)-kke 67 552807 ekk-d(10)-kke 61 552808 ekk-d(10)-kke 83 552809 ekk-d(10)-kke 59 552810 ekk-d(10)-kke 56 552811 ekk-d(10)-kke 62 552812 ekk-d(10)-kke 66 552813 ekk-d(10)-kke 63 552814 ekk-d(10)-kke 65 552815 ekk-d(10)-kke 63 552816 ekk-d(10)-kke 88 552817 ekk-d(10)-kke 94 552818 ekk-d(10)-kke 82 552819 ekk-d(10)-kke 80 552820 ekk-d(10)-kke 84 552821 ekk-d(10)-kke 71 552822 ekk-d(10)-kke 85 552823 ekk-d(10)-kke 71 552824 ekk-d(10)-kke 81 552825 ekk-d(10)-kke 51 552826 ekk-d(10)-kke 64 552827 ekk-d(10)-kke 61 552828 ekk-d(10)-kke 76 552829 ekk-d(10)-kke 61 552830 ekk-d(10)-kke 59 552831 ekk-d(10)-kke 58 552832 ekk-d(10)-kke 64 552833 ekk-d(10)-kke 75 552834 ekk-d(10)-kke 84 552835 ekk-d(10)-kke 57 552836 ekk-d(10)-kke 51 552837 ekk-d(10)-kke 53 552838 ekk-d(10)-kke 48 552839 ekk-d(10)-kke 50 552840 ekk-d(10)-kke 54 552841 ekk-d(10)-kke 61 552842 ekk-d(10)-kke 71 552843 ekk-d(10)-kke 75 552844 ekk-d(10)-kke 78 552845 ekk-d(10)-kke 52 552846 ekk-d(10)-kke 76 552847 ekk-d(10)-kke 61 552848 ekk-d(10)-kke 72 552849 ekk-d(10)-kke 87 552850 ekk-d(10)-kke 76 552851 ekk-d(10)-kke 76 552852 ekk-d(10)-kke 79 552853 ekk-d(10)-kke 82 552854 ekk-d(10)-kke 85 552855 ekk-d(10)-kke 78 552856 ekk-d(10)-kke 77 552857 ekk-d(10)-kke 75 552858 ekk-d(10)-kke 75 552859 ekk-d(10)-kke 79 552860 ekk-d(10)-kke 71 552861 ekk-d(10)-kke 74 552862 ekk-d(10)-kke 66 552863 ekk-d(10)-kke 70 552864 ekk-d(10)-kke 73 e = 2'-MOE, k = cEt, d = 2'-deoxynucleoside

TABLE 66 Inhibition of viral Target-Z mRNA levels by chimeric antisense oligonucleotidesmeasured with RTS3371 % ISIS No Motif inhibition 146786 eeeee-d(10)-eeeee 60 552889 ek-d(10)-keke 59 552890 ek-d(10)-keke 56 552891 ek-d(10)-keke 67 552892 ek-d(10)-keke 65 552893 ek-d(10)-keke 68 552894 ek-d(10)-keke 71 552895 ek-d(10)-keke 51 552896 ek-d(10)-keke 51 552897 ek-d(10)-keke 43 552898 ek-d(10)-keke 43 552899 ek-d(10)-keke 55 552900 ek-d(10)-keke 34 552901 ek-d(10)-keke 42 552902 ek-d(10)-keke 60 552903 ek-d(10)-keke 76 552904 ek-d(10)-keke 74 552905 ek-d(10)-keke 66 552907 ek-d(10)-keke 69 552908 ek-d(10)-keke 63 552909 ek-d(10)-keke 70 552910 ek-d(10)-keke 72 552911 ek-d(10)-keke 72 552912 ek-d(10)-keke 67 552913 ek-d(10)-keke 74 552914 ek-d(10)-keke 75 552915 ek-d(10)-keke 58 552916 ek-d(10)-keke 74 552917 ek-d(10)-keke 76 552918 ek-d(10)-keke 75 552919 ek-d(10)-keke 55 552920 ek-d(10)-keke 49 552921 ek-d(10)-keke 45 552922 ek-d(10)-keke 83 552923 ek-d(10)-keke 83 552924 ek-d(10)-keke 0 552925 ek-d(10)-keke 85 552926 ek-d(10)-keke 50 552927 ek-d(10)-keke 76 552928 ek-d(10)-keke 78 552929 ek-d(10)-keke 75 552930 ek-d(10)-keke 78 552931 ek-d(10)-keke 74 552932 ek-d(10)-keke 86 552933 ek-d(10)-keke 82 552934 ek-d(10)-keke 74 552935 ek-d(10)-keke 76 552936 ek-d(10)-keke 81 552937 ek-d(10)-keke 80 552938 ek-d(10)-keke 78 552939 ek-d(10)-keke 75 552940 ek-d(10)-keke 63 552941 ekk-d(10)-kke 78 552942 ek-d(10)-keke 80 552865 ekk-d(10)-kke 67 552866 ekk-d(10)-kke 68 552868 ekk-d(10)-kke 55 552869 ekk-d(10)-kke 48 552870 ekk-d(10)-kke 55 552871 ekk-d(10)-kke 57 552872 ekk-d(10)-kke 70 552873 ekk-d(10)-kke 49 552874 ekk-d(10)-kke 42 552875 ekk-d(10)-kke 41 552876 ekk-d(10)-kke 50 552877 ek-d(10)-keke 39 552878 ekk-d(10)-kke 31 552879 ekk-d(10)-kke 5 552880 ekk-d(10)-kke 5 552881 ekk-d(10)-kke 10 552882 ekk-d(10)-kke 11 552883 ekk-d(10)-kke 27 552884 ekk-d(10)-kke 36 552885 ekk-d(10)-kke 12 552886 ekk-d(10)-kke 32 552888 ekk-d(10)-kke 1 e = 2'-MOE, k = cEt, d = 2'-deoxynucleoside

TABLE 67 Inhibition of viral Target-Z mRNA levels by chimeric antisense oligonucleotidesmeasured with RTS3371 % ISIS No Motif inhibition 146786 eeeee-d(10)-eeeee 59 552955 eee-d(10)-kkk 60 552956 eee-d(10)-kkk 60 552957 eee-d(10)-kkk 64 552958 eee-d(10)-kkk 56 552959 eee-d(10)-kkk 59 552960 eee-d(10)-kkk 42 552961 eee-d(10)-kkk 41 552962 eee-d(10)-kkk 35 552963 eee-d(10)-kkk 19 552964 eee-d(10)-kkk 34 552965 eee-d(10)-kkk 42 552966 eee-d(10)-kkk 60 552967 eee-d(10)-kkk 38 552968 eee-d(10)-kkk 35 552969 eee-d(10)-kkk 67 552970 eee-d(10)-kkk 56 552971 eee-d(10)-kkk 69 552972 eee-d(10)-kkk 75 552973 eee-d(10)-kkk 59 552974 eee-d(10)-kkk 71 552975 eee-d(10)-kkk 56 552976 eee-d(10)-kkk 50 552977 eee-d(10)-kkk 56 552978 eee-d(10)-kkk 43 552979 eee-d(10)-kkk 71 552980 eee-d(10)-kkk 80 552981 eee-d(10)-kkk 64 552982 ek-d(10)-keke 61 552983 eee-d(10)-kkk 77 552984 eee-d(10)-kkk 65 552985 eee-d(10)-kkk 41 552986 eee-d(10)-kkk 30 552987 eee-d(10)-kkk 41 552988 eee-d(10)-kkk 74 552989 eee-d(10)-kkk 85 552990 eee-d(10)-kkk 72 552991 eee-d(10)-kkk 73 552992 eee-d(10)-kkk 60 552993 eee-d(10)-kkk 52 552994 eee-d(10)-kkk 58 552995 eee-d(10)-kkk 70 552996 eee-d(10)-kkk 74 552997 eee-d(10)-kkk 59 552998 eee-d(10)-kkk 82 552999 eee-d(10)-kkk 70 553000 eee-d(10)-kkk 67 553001 eee-d(10)-kkk 67 553002 eee-d(10)-kkk 74 553003 eee-d(10)-kkk 72 553004 eee-d(10)-kkk 73 553005 eee-d(10)-kkk 67 553006 eee-d(10)-kkk 69 553007 eee-d(10)-kkk 60 553008 eee-d(10)-kkk 71 552943 ek-d(10)-keke 77 553009 eee-d(10)-kkk 78 552944 ek-d(10)-keke 74 553010 eee-d(10)-kkk 78 552945 ek-d(10)-keke 76 553011 eee-d(10)-kkk 72 552946 ek-d(10)-keke 71 553012 eee-d(10)-kkk 74 552947 ek-d(10)-keke 54 553013 eee-d(10)-kkk 39 552948 ek-d(10)-keke 50 553014 eee-d(10)-kkk 37 552949 ek-d(10)-keke 8 553015 eee-d(10)-kkk 45 552950 ek-d(10)-keke 44 553016 eee-d(10)-kkk 47 552951 ek-d(10)-keke 60 553017 eee-d(10)-kkk 47 552952 ek-d(10)-keke 35 553018 eee-d(10)-kkk 30 552953 ek-d(10)-keke 37 553019 eee-d(10)-kkk 37 552954 ek-d(10)-keke 40 553020 eee-d(10)-kkk 24 e = 2'-MOE, k = cEt, d = 2'-deoxynucleoside

TABLE 68 Inhibition of viral Target-Z mRNA levels by chimeric antisense oligonucleotidesmeasured with RTS3370 % ISIS No Motif inhibition 552889 ek-d(10)-keke 42 552890 ek-d(10)-keke 56 552891 ek-d(10)-keke 55 552892 ek-d(10)-keke 53 552893 ek-d(10)-keke 56 552894 ek-d(10)-keke 53 552895 ek-d(10)-keke 38 552896 ek-d(10)-keke 43 552897 ek-d(10)-keke 40 552898 ek-d(10)-keke 50 552899 ek-d(10)-keke 37 552900 ek-d(10)-keke 43 552901 ek-d(10)-keke 56 552902 ek-d(10)-keke 43 552903 ek-d(10)-keke 78 552904 ek-d(10)-keke 75 552905 ek-d(10)-keke 52 552907 ek-d(10)-keke 75 552908 ek-d(10)-keke 57 552909 ek-d(10)-keke 66 552910 ek-d(10)-keke 60 552911 ek-d(10)-keke 65 552912 ek-d(10)-keke 37 552913 ek-d(10)-keke 76 552914 ek-d(10)-keke 79 552915 ek-d(10)-keke 71 552916 ek-d(10)-keke 82 552917 ek-d(10)-keke 78 552918 ek-d(10)-keke 64 552919 ek-d(10)-keke 38 552920 ek-d(10)-keke 43 552921 ek-d(10)-keke 49 552922 ek-d(10)-keke 90 552923 ek-d(10)-keke 92 552924 ek-d(10)-keke 30 552925 ek-d(10)-keke 81 552926 ek-d(10)-keke 39 552927 ek-d(10)-keke 53 552928 ek-d(10)-keke 48 552929 ek-d(10)-keke 68 552930 ek-d(10)-keke 87 552931 ek-d(10)-keke 87 552932 ek-d(10)-keke 88 552933 ek-d(10)-keke 75 552934 ek-d(10)-keke 76 552935 ek-d(10)-keke 71 552936 ek-d(10)-keke 80 552937 ek-d(10)-keke 81 552938 ek-d(10)-keke 85 552939 ek-d(10)-keke 82 552940 ek-d(10)-keke 76 552941 ekk-d(10)-kke 72 552942 ek-d(10)-keke 85 552865 ekk-d(10)-kke 70 552866 ekk-d(10)-kke 65 552868 ekk-d(10)-kke 36 552869 ekk-d(10)-kke 23 552870 ekk-d(10)-kke 49 552871 ekk-d(10)-kke 46 552872 ekk-d(10)-kke 73 552873 ekk-d(10)-kke 41 552874 ekk-d(10)-kke 18 552875 ekk-d(10)-kke 0 552876 ekk-d(10)-kke 49 552877 ek-d(10)-keke 37 552878 ekk-d(10)-kke 28 552879 ekk-d(10)-kke 0 552880 ekk-d(10)-kke 12 552881 ekk-d(10)-kke 0 552882 ekk-d(10)-kke 0 552883 ekk-d(10)-kke 12 552884 ekk-d(10)-kke 39 552885 ekk-d(10)-kke 37 552886 ekk-d(10)-kke 15 552888 ekk-d(10)-kke 0 e = 2'-MOE, k = cEt, d = 2'-deoxynucleoside

TABLE 69 Inhibition of viral Target-Z mRNA levels by chimeric antisense oligonucleotidesmeasured with RTS3370 % ISIS No Motif inhibition 552955 eee-d(10)-kkk 67 552956 eee-d(10)-kkk 60 552957 eee-d(10)-kkk 73 552958 eee-d(10)-kkk 63 552959 eee-d(10)-kkk 58 552960 eee-d(10)-kkk 67 552961 eee-d(10)-kkk 78 552962 eee-d(10)-kkk 29 552963 eee-d(10)-kkk 25 552964 eee-d(10)-kkk 33 552965 eee-d(10)-kkk 55 552966 eee-d(10)-kkk 71 552967 eee-d(10)-kkk 23 552968 eee-d(10)-kkk 41 552969 eee-d(10)-kkk 76 552970 eee-d(10)-kkk 44 552971 eee-d(10)-kkk 77 552972 eee-d(10)-kkk 74 552973 eee-d(10)-kkk 61 552974 eee-d(10)-kkk 73 552975 eee-d(10)-kkk 66 552976 eee-d(10)-kkk 70 552977 eee-d(10)-kkk 65 552978 eee-d(10)-kkk 40 552979 eee-d(10)-kkk 79 552980 eee-d(10)-kkk 81 552981 eee-d(10)-kkk 74 552982 ek-d(10)-keke 52 552983 eee-d(10)-kkk 78 552984 eee-d(10)-kkk 71 552985 eee-d(10)-kkk 38 552986 eee-d(10)-kkk 48 552987 eee-d(10)-kkk 54 552988 eee-d(10)-kkk 85 552989 eee-d(10)-kkk 84 552990 eee-d(10)-kkk 79 552991 eee-d(10)-kkk 53 552992 eee-d(10)-kkk 68 552993 eee-d(10)-kkk 67 552994 eee-d(10)-kkk 69 552995 eee-d(10)-kkk 62 552996 eee-d(10)-kkk 82 552997 eee-d(10)-kkk 58 552998 eee-d(10)-kkk 86 552999 eee-d(10)-kkk 63 553000 eee-d(10)-kkk 67 553001 eee-d(10)-kkk 70 553002 eee-d(10)-kkk 84 553003 eee-d(10)-kkk 83 553004 eee-d(10)-kkk 68 553005 eee-d(10)-kkk 57 553006 eee-d(10)-kkk 74 553007 eee-d(10)-kkk 62 553008 eee-d(10)-kkk 50 552943 ek-d(10)-keke 86 553009 eee-d(10)-kkk 79 552944 ek-d(10)-keke 83 553010 eee-d(10)-kkk 74 552945 ek-d(10)-keke 79 553011 eee-d(10)-kkk 60 552946 ek-d(10)-keke 68 553012 eee-d(10)-kkk 78 552947 ek-d(10)-keke 51 553013 eee-d(10)-kkk 45 552948 ek-d(10)-keke 56 553014 eee-d(10)-kkk 53 552949 ek-d(10)-keke 1 553015 eee-d(10)-kkk 55 552950 ek-d(10)-keke 52 553016 eee-d(10)-kkk 65 552951 ek-d(10)-keke 59 553017 eee-d(10)-kkk 36 552952 ek-d(10)-keke 34 553018 eee-d(10)-kkk 20 552953 ek-d(10)-keke 55 553019 eee-d(10)-kkk 34 552954 ek-d(10)-keke 51 553020 eee-d(10)-kkk 28 e = 2'-MOE, k = cEt, d = 2'-deoxynucleoside

Example 47 Dose-Dependent Antisense Inhibition of Target-Z mRNA in HepG2 Cells

Antisense oligonucleotides from the study described in Example 46 exhibiting in vitro inhibition of Target-Z mRNA were selected and tested at various doses in HepG2 cells. Cells were plated at a density of 28,000 cells per well and transfected using LipofectAMINE2000® with 9.26 nM, 27.78 nM, 83.33 nM, and 250.00 nM concentrations of antisense oligonucleotide, as specified in Table 70. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-Z mRNA levels were measured by quantitative real-time PCR. Target-Z primer probe set RTS3371 was used to measure mRNA levels. Target-Z mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-Z, relative to untreated control cells.

As illustrated in Table 70, Target-Z mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 70 Dose-dependent antisense inhibition of human Target-Z in HepG2 cells 9.2593 27.7778 83.3333 250.0 ISIS No Motif nM nM nM nM 146786 eeeee-d(10)-eeeee 10 43 74 89 552808 ekk-d(10)-kke 13 14 55 70 552816 ekk-d(10)-kke 38 73 87 92 552818 ekk-d(10)-kke 29 63 87 85 552820 ekk-d(10)-kke 58 83 90 90 552821 ekk-d(10)-kke 33 49 71 88 552822 ekk-d(10)-kke 24 55 74 88 552824 ekk-d(10)-kke 8 24 65 87 552834 ekk-d(10)-kke 11 28 68 89 552849 ekk-d(10)-kke 12 25 73 84 552851 ekk-d(10)-kke 13 42 74 89 552852 ekk-d(10)-kke 4 35 70 87 552853 ekk-d(10)-kke 19 52 86 93 552854 ekk-d(10)-kke 28 57 80 89 552916 ek-d(10)-keke 5 26 64 82 552922 ek-d(10)-keke 25 44 77 89 552923 ek-d(10)-keke 22 49 82 91 552925 ek-d(10)-keke 33 56 80 92 552930 ek-d(10)-keke 12 49 79 89 552931 ek-d(10)-keke 12 40 62 82 552932 ek-d(10)-keke 24 62 84 91 552933 ek-d(10)-keke 20 40 75 89 552936 ek-d(10)-keke 18 36 75 88 552937 ek-d(10)-keke 22 51 82 88 552938 ek-d(10)-keke 12 36 67 80 552939 ek-d(10)-keke 17 40 65 79 552942 ek-d(10)-keke 21 48 74 88 552943 ek-d(10)-keke 5 39 70 85 552944 ek-d(10)-keke 14 33 70 77 552980 eee-d(10)-kkk 15 40 69 86 552988 eee-d(10)-kkk 4 36 58 84 552989 eee-d(10)-kkk 0 50 74 81 552996 eee-d(10)-kkk 0 25 53 72 552998 eee-d(10)-kkk 17 49 79 90 553002 eee-d(10)-kkk 0 32 68 86 553003 eee-d(10)-kkk 15 42 67 88 e = 2'-MOE, k = cEt, d = 2'-deoxynucleoside

Example 48 Efficacy of Antisense Oligonucleotides Targeting Target-Z in Transgenic Mice

Mice harboring a Target-Z gene fragment (Guidotti, L. G. et al., J. Virol. 1995, 69, 6158-6169) were used. The mice were treated with ISIS antisense oligonucleotides selected from studies described above as illustrated in Table 71 and evaluated for their efficacy in this model.

Treatment

Groups of 10 mice each were injected subcutaneously twice a week for the first with 50 mg/kg and, subsequently, twice a week for the next 3 weeks with 25 mg/kg of ISIS 146786 or ISIS 510100. Control groups of 10 mice each were treated in a similar manner with ISIS 141923 (5-10-5 MOE gapmer with no known murine target) or ISIS 459024 (3-10-4 MOE gapmer with no known murine target). Mice were euthanized 48 hours after the last dose, and organs and serum were harvested for further analysis.

TABLE 71 Antisense oligonucleotides targeting Target-Z in transgenic mice ISIS No. Sequence (5′ to 3′) Motif SEQ ID NO. 146786 G_(es)T_(es)G_(es)aA_(es)A_(es)G_(ds)C_(ds)G_(ds)A_(ds)A_(ds) e5-d(10)-e5 39 Ga_(s)T_(ds)G_(ds)C_(ds)A_(ds)C_(es)A_(es)C_(es)G_(es)G_(es) 510100 G_(es)G_(es) ^(m)C_(es)A_(ds)T_(ds)A_(ds)G_(ds) ^(m)C_(ds)A_(ds) eee-d(10)-eeee 40 G_(ds) ^(m)C_(ds)A_(ds)G_(ds)G_(es)A_(es)T_(es)G_(e) 141923 ^(m)C_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es) ^(m)C_(ds) ^(m)C_(ds)T_(ds)G_(ds)A_(ds) e5-d(10)-e5 41 A_(ds)G_(ds)G_(ds)T_(ds)T_(ds) ^(m)C_(es) ^(m)C_(es)T_(es) ^(m)C_(es) ^(m)C_(e) 459024 ^(m)C_(es)G_(es)G_(es)T_(ds) ^(m)C_(ds) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)G_(ds) eee-d(10)-eeee 42 A_(ds)G_(ds)G_(ds)A_(es)T_(es)G_(es) ^(m)C_(e) e = 2′-MOE (e.g. e5 = eeeee), d = 2′-deoxynucleoside

DNA and RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of Target-Z DNA, using primer probe sets RTS3370, RTS3371, or RTS3372 (forward sequence ATCCTATCAACACTTCCGGAAACT, designated SEQ ID NO: 43; reverse sequence CGACGCGGCGATTGAG, designated SEQ ID NO: 44; probe sequence AAGAACTCCCTCGCCTCGCAGACG, designated SEQ ID NO: 45). The DNA levels were normalized to picogreen. Target-Z RNA samples were also assayed with primer probe sets RTS3370 and RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. The data is presented in Table 72. Serum DNA samples were analyzed after the study period. The data is presented in Table 73, expressed relative to the levels measured in the control group. As shown in Tables 72 and 73, the antisense oligonucleotides achieved reduction of Target-Z DNA and RNA over the PBS control. Treatment with either control oligonucleotide did not cause any changes in RNA or DNA levels, as expected.

TABLE 72 Percent inhibition of Target-Z RNA and DNA in the liver of transgenic mice % % % % % % inhibition inhibition inhibition inhibition inhibition inhibition DNA DNA DNA RNA RNA RNA ISIS No Motif (RTS3370) (RTS3371) (RTS3372) (RTS3370) (RTS3371) (RTS3372) 146786 e5-d(10)-e5 97 97 95 86 85 89 510100 eee-d(10)-eeee 95 94 94 56 64 77 141923 e5-d(10)-e5 2 0 13 0 7 31 459024 eee-d(10)-eeee 19 0 8 0 0 0 e = 2′-MOE (e.g. e5 = eeeee), d = 2′-deoxynucleoside

TABLE 73 Percent inhibition of Target-Z DNA in the serum of transgenic mice % inhibition % inhibition ISIS No Motif (RTS3370) (RTS3371) 146786 e5-d(10)-e5 98 98 510100 eee-d(10)-eeee 99 98 141923 e5-d(10)-e5 0 0 459024 eee-d(10)-eeee 0 0 e = 2'-MOE (e.g. e5 = eeeee), d = 2'-deoxynucleoside

Example 49 Efficacy of Antisense Oligonucleotides Targeting Target-Z in Transgenic Mice

Transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for their efficacy in this model.

Treatment

A group of 6 mice was injected subcutaneously twice a week for 4 weeks with 25 mg/kg of ISIS 146786. Groups of 6 mice each were injected subcutaneously twice a week for 4 weeks with 10 mg/kg of ISIS 552803, ISIS 552903, ISIS 552817, ISIS 552822, and ISIS 552907. One group of 10 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

DNA and RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of Target-Z DNA, using primer probe set RTS3371. The DNA levels were normalized to picogreen. Target-Z RNA samples were also assayed with primer probe set RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. The data is presented in Table 74. Serum DNA samples were analyzed after the study period. The data is presented in Table 75, expressed relative to the levels measured in the control group. As shown in Tables 74 and 75, the antisense oligonucleotides achieved reduction of Target-Z DNA and RNA over the PBS control.

TABLE 74 Percent inhibition of Target-Z RNA and DNA in transgenic mice % % Dose inhibition inhibition ISIS No Motif (mg/kg/wk) of RNA of DNA 146786 e5-d(10)-e5 50 81 91 552803 ekk-d(10)-kke 20 71 95 552817 ekk-d(10)-kke 20 86 51 552822 ekk-d(10)-kke 20 90 89 552903 ek-d(10)-keke 20 56 82 552907 ek-d(10)-keke 20 41 45 e = 2'-MOE (e.g. e5 = eeeee). d = 2'-deoxynucleoside

TABLE 75 Serum levels of Target-Z DNA in transgenic mice, relative to control levels Post-dose Dose DNA ISIS No Motif (mg/kg/wk) levels 146786 e5-d(10)-e5 50 0.1 552803 ekk-d(10)-kke 20 0.2 552817 ekk-d(10)-kke 20 1.3 552822 ekk-d(10)-kke 20 0.0 552903 ek-d(10)-keke 20 2.9 552907 ek-d(10)-keke 20 1.0 e = 2'-MOE (e.g. e5 = eeeee), d = 2'-deoxynucleoside

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma concentrations of ALT were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.) (Nyblom, H. et al., Alcohol & Alcoholism 39: 336-339, 2004; Tietz N W (Ed): Clinical Guide to Laboratory Tests, 3rd ed. W. B. Saunders, Philadelphia, Pa., 1995). The results are presented in Table 76 expressed in IU/L. All the ISIS oligonucleotides were considered tolerable in the mice, as demonstrated by their liver transaminase profile.

TABLE 76 ALT levels (IU/L) of transgenic mice Dose Motif (mg/kg/wk) ALT PBS — — 77 ISIS 146786 e5-d(10)-e5 50 21 ISIS 552803 ekk-d(10)-kke 20 74 ISIS 552817 ekk-d(10)-kke 20 38 ISIS 552822 ekk-d(10)-kke 20 47 ISIS 552903 ek-d(10)-keke 20 57 ISIS 552907 ek-d(10)-keke 20 28 e = 2'-MOE (e.g. e5 = eeeee), d = 2'-deoxynucleoside

Example 50 Efficacy of Antisense Oligonucleotides Targeting Target-Z in Transgenic Mice

Transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for their efficacy in this model.

Treatment

A group of 6 mice was injected subcutaneously twice a week for 4 weeks with 25 mg/kg of ISIS 146786. Groups of 6 mice each were injected subcutaneously twice a week for 4 weeks with 10 mg/kg of ISIS 552853, ISIS 552854, ISIS 552932, and ISIS 552938. One group of 10 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

DNA and RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of Target-Z DNA, using primer probe set RTS3371. The DNA levels were normalized to picogreen. Target-Z RNA samples were also assayed with primer probe set RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. As shown in Table 77, the antisense oligonucleotides achieved reduction of Target-Z DNA and RNA over the PBS control. Results are presented as percent inhibition of Target-Z mRNA or DNA, relative to control.

TABLE 77 Percent inhibition of Target-Z DNA and RNA in transgenic mice % % Dose inhibition inhibition Motif (mg/kg/wk) (DNA) (RNA) PBS — — ISIS 146786 e5-d(10)-e5 50 90 60 ISIS 552853 ekk-d(10)-kke 20 94 60 ISIS 552854 ekk-d(10)-kke 20 61 23 ISIS 552932 ekk-d(10)-kke 20 75 70 ISIS 552938 ek-d(10)-keke 20 67 56 = 2'-MOE (e.g. e5 = eeeee), d = 2'-deoxynucleoside

Example 51 Efficacy of Antisense Oligonucleotides Targeting Target-Z in Transgenic Mice

Transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for their efficacy in this model.

Treatment

A group of 6 mice was injected subcutaneously twice a week for 4 weeks with 25 mg/kg of ISIS 146786. Groups of 6 mice each were injected subcutaneously twice a week for 4 weeks with 10 mg/kg of ISIS 552922, ISIS 552923, ISIS 552942, ISIS 552872, ISIS 552925, ISIS 552937, and ISIS 552939. One group of 10 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

DNA and RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of Target-Z DNA, using primer probe set RTS3371. The DNA levels were normalized to picogreen. Target-Z RNA samples were also assayed with primer probe set RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. As shown in Table 78, the antisense oligonucleotides achieved reduction of Target-Z DNA and RNA over the PBS control. Results are presented as percent inhibition of Target-Z mRNA or DNA, relative to control.

TABLE 78 Percent inhibition of Target-Z DNA and RNA in transgenic mice % % Dose inhibition inhibition ISIS No Motif (mg/kg/wk) (DNA) (RNA) 146786 e5-d(10)-e5 50 52 57 552922 ek-d(10)-keke 20 61 50 552923 ek-d(10)-keke 20 89 76 552942 ek-d(10)-keke 20 58 52 552872 ekk-d(10)-keke 20 77 46 552925 ek-d(10)-keke 20 89 65 552937 ek-d(10)-keke 20 59 35 552939 ek-d(10)-keke 20 57 19 = 2'-MOE (e.g. e5 = eeeee), d = 2'-deoxynucleoside

Example 52 Antisense Inhibition of Target-Z mRNA in HepG2 Cells

Antisense oligonucleotides were designed targeting a Target-Z nucleic acid and were tested for their effects on Target-Z mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables. ISIS 146786, 509934, ISIS 509959, and ISIS 510100, from the studies described above, were also included. Cultured HepG2 cells at a density of 28,000 cells per well were transfected using LipofectAMINE2000® with 70 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-Z mRNA levels were measured by quantitative real-time PCR. Primer probe set RTS3370 (forward sequence CTTGGTCATGGGCCATCAG, designated herein as SEQ ID NO: 33; reverse sequence CGGCTAGGAGTTCCGCAGTA, designated herein as SEQ ID NO: 34; probe sequence TGCGTGGAACCTTTTCGGCTCC, designated herein as SEQ ID NO: 35) was used to measure mRNA levels. Levels were also measured using primer probe set RTS3371 (forward sequence CCAAACCTTCGGACGGAAA, designated herein as SEQ ID NO: 36; reverse sequence TGAGGCCCACTCCCATAGG, designated herein as SEQ ID NO: 37; probe sequence CCCATCATCCTGGGCTTTCGGAAAAT, designated herein as SEQ ID NO: 38). Target-Z mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-Z, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides and their motifs are described in Tables 79-96. The modified oligonucleotides are 16, 17 or 20 nucleotides in length, wherein the central gap segment comprises of nine or ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising 2′-O-methoxyethyl (2′-MOE) modifications. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.

Each gapmer listed in the Tables is targeted to the viral genomic sequence, designated herein as Target-Z. The activity of the newly designed oligonucleotides was compared with ISIS 146786, 509934, ISIS 509959, and ISIS 510100, the information of which have been placed at the top of each table.

TABLE 79 Inhibition of viral Target-Z mRNA levels by chimeric antisense oligonucleotidesmeasured with RTS3370 Wing % ISIS No Motif chemistry inhibition 146786 5-10-5 2'-MOE 50 510100 3-10-4 2'-MOE 62 552276 5-9-3 2'-MOE 42 552277 5-9-3 2'-MOE 46 552278 5-9-3 2'-MOE 31 552279 5-9-3 2'-MOE 41 552280 5-9-3 2'-MOE 5 552281 5-9-3 2'-MOE 11 552282 5-9-3 2'-MOE 20 552283 5-9-3 2'-MOE 28 552230 4-9-4 2'-MOE 57 552284 5-9-3 2'-MOE 0 552231 4-9-4 2'-MOE 29 552285 5-9-3 2'-MOE 61 552232 4-9-4 2'-MOE 35 552286 5-9-3 2'-MOE 47 552233 4-9-4 2'-MOE 38 552287 5-9-3 2'-MOE 45 552234 4-9-4 2'-MOE 0 552288 5-9-3 2'-MOE 50 552235 4-9-4 2'-MOE 0 552289 5-9-3 2'-MOE 46 552236 4-9-4 2'-MOE 45 552290 5-9-3 2'-MOE 41 552237 4-9-4 2'-MOE 44 552291 5-9-3 2'-MOE 26 552239 4-9-4 2'-MOE 62 552293 5-9-3 2'-MOE 67 552240 4-9-4 2'-MOE 61 552294 5-9-3 2'-MOE 71 552241 4-9-4 2'-MOE 55 552295 5-9-3 2'-MOE 58 552242 4-9-4 2'-MOE 60 552296 5-9-3 2'-MOE 59 552243 4-9-4 2'-MOE 57 552297 5-9-3 2'-MOE 55 552244 4-9-4 2'-MOE 33 552298 5-9-3 2'-MOE 48 552245 4-9-4 2'-MOE 48 552299 5-9-3 2'-MOE 34 552246 4-9-4 2'-MOE 81 552300 5-9-3 2'-MOE 56 552247 4-9-4 2'-MOE 87 552301 5-9-3 2'-MOE 86 552248 4-9-4 2'-MOE 72 552302 5-9-3 2'-MOE 77 552249 4-9-4 2'-MOE 56 552303 5-9-3 2'-MOE 65 552250 4-9-4 2'-MOE 52 552304 5-9-3 2'-MOE 57 552251 4-9-4 2'-MOE 43 552305 5-9-3 2'-MOE 56 552252 4-9-4 2'-MOE 62 552306 5-9-3 2'-MOE 75 552253 4-9-4 2'-MOE 82 552307 5-9-3 2'-MOE 90 552254 4-9-4 2'-MOE 74 552255 4-9-4 2'-MOE 78 552256 4-9-4 2'-MOE 65 552257 4-9-4 2'-MOE 62 552258 4-9-4 2'-MOE 72 552259 4-9-4 2'-MOE 63 552260 4-9-4 2'-MOE 58 552261 4-9-4 2'-MOE 63 552262 4-9-4 2'-MOE 50 552263 4-9-4 2'-MOE 60 552264 4-9-4 2'-MOE 52 552265 4-9-4 2'-MOE 68 552266 4-9-4 2'-MOE 62 552267 4-9-4 2'-MOE 58 552268 4-9-4 2'-MOE 62 552269 4-9-4 2'-MOE 52 552270 4-9-4 2'-MOE 54 552271 4-9-4 2'-MOE 58 552272 4-9-4 2'-MOE 40 552273 4-9-4 2'-MOE 34 552274 4-9-4 2'-MOE 34 552275 4-9-4 2'-MOE 39

TABLE 80 Inhibition of viral Target-Z mRNA levels by chimeric antisense oligonucleotidesmeasured with RTS3370 Wing % ISIS No Motif chemistry inhibition 146786 5-10-5 2'-MOE 49 509959 3-10-3 2'-MOE 43 510100 3-10-4 2'-MOE 54 552384 2-9-5 2'-MOE 29 552440 3-9-4 2'-MOE 58 552385 2-9-5 2'-MOE 57 552441 3-9-4 2'-MOE 42 552386 2-9-5 2'-MOE 53 552442 3-9-4 2'-MOE 53 552387 2-9-5 2'-MOE 48 552443 3-9-4 2'-MOE 59 552388 2-9-5 2'-MOE 40 552444 3-9-4 2'-MOE 51 552389 2-9-5 2'-MOE 39 552445 3-9-4 2'-MOE 60 552390 2-9-5 2'-MOE 52 552446 3-9-4 2'-MOE 54 552391 2-9-5 2'-MOE 57 552/147 3-9-4 2'-MOE 54 552392 2-9-5 2'-MOE 0 552448 3-9-4 2'-MOE 58 552393 2-9-5 2'-MOE 59 552449 3-9-4 2'-MOE 60 552394 2-9-5 2'-MOE 53 552450 3-9-4 2'-MOE 53 552395 2-9-5 2'-MOE 57 552451 3-9-4 2'-MOE 39 552396 2-9-5 2'-MOE 62 552452 3-9-4 2'-MOE 57 552238 4-9-4 2'-MOE 38 552292 5-9-3 2'-MOE 48 552346 6-9-2 2'-MOE 0 552397 2-9-5 2'-MOE 63 552453 3-9-4 2'-MOE 56 552398 2-9-5 2'-MOE 61 552454 3-9-4 2'-MOE 48 552399 2-9-5 2'-MOE 52 552400 2-9-5 2'-MOE 57 552401 2-9-5 2'-MOE 52 552402 2-9-5 2'-MOE 54 552403 2-9-5 2'-MOE 74 552404 2-9-5 2'-MOE 43 552405 2-9-5 2'-MOE 15 552406 2-9-5 2'-MOE 37 552407 2-9-5 2'-MOE 37 552408 2-9-5 2'-MOE 76 552409 2-9-5 2'-MOE 76 552410 2-9-5 2'-MOE 63 552411 2-9-5 2'-MOE 70 552412 2-9-5 2'-MOE 62 552413 2-9-5 2'-MOE 56 552414 2-9-5 2'-MOE 63 552415 2-9-5 2'-MOE 52 552416 2-9-5 2'-MOE 67 552417 2-9-5 2'-MOE 50 552418 2-9-5 2'-MOE 79 552419 2-9-5 2'-MOE 70 552420 2-9-5 2'-MOE 71 552421 2-9-5 2'-MOE 69 552422 2-9-5 2'-MOE 68 552423 2-9-5 2'-MOE 65 552424 2-9-5 2'-MOE 70 552425 2-9-5 2'-MOE 51 552426 2-9-5 2'-MOE 40 552427 2-9-5 2'-MOE 35 552428 2-9-5 2'-MOE 58 552429 2-9-5 2'-MOE 46 552430 2-9-5 2'-MOE 53 552431 2-9-5 2'-MOE 51 552432 2-9-5 2'-MOE 57 552433 2-9-5 2'-MOE 54 552434 2-9-5 2'-MOE 44 552435 2-9-5 2'-MOE 46 552436 2-9-5 2'-MOE 36 552437 2-9-5 2'-MOE 27 552438 2-9-5 2'-MOE 27 552439 2-9-5 2'-MOE 13

TABLE 81 Inhibition of viral Target-Z mRNA levels by chimeric antisense oligonucleotidesmeasurewith RTS3370 Wing % ISIS No Motif chemistry inhibition 146786 5-10-5 2'-MOE 35 509959 3-10-3 2'-MOE 52 552496 4-9-3 2'-MOE 47 552497 4-9-3 2'-MOE 57 552498 4-9-3 2'-MOE 45 552499 4-9-3 2'-MOE 52 552500 4-9-3 2'-MOE 46 552501 4-9-3 2'-MOE 44 552502 4-9-3 2'-MOE 57 552503 4-9-3 2'-MOE 52 552504 4-9-3 2'-MOE 45 552505 4-9-3 2'-MOE 56 552506 4-9-3 2'-MOE 54 552507 4-9-3 2'-MOE 34 552508 4-9-3 2'-MOE 34 552509 4-9-3 2'-MOE 48 552510 4-9-3 2'-MOE 50 552455 3-9-4 2'-MOE 66 552511 4-9-3 2'-MOE 66 552456 3-9-4 2'-MOE 64 552512 4-9-3 2'-MOE 62 552457 3-9-4 2'-MOE 14 552513 4-9-3 2'-MOE 56 552458 3-9-4 2'-MOE 59 552514 4-9-3 2'-MOE 52 552459 3-9-4 2'-MOE 69 552515 4-9-3 2'-MOE 57 552460 3-9-4 2'-MOE 0 552516 4-9-3 2'-MOE 54 552461 3-9-4 2'-MOE 20 552517 4-9-3 2'-MOE 52 552462 3-9-4 2'-MOE 46 552518 4-9-3 2'-MOE 34 552463 3-9-4 2'-MOE 48 552519 4-9-3 2'-MOE 44 552464 3-9-4 2'-MOE 81 552520 4-9-3 2'-MOE 69 552465 3-9-4 2'-MOE 84 552521 4-9-3 2'-MOE 80 552466 3-9-4 2'-MOE 75 552522 4-9-3 2'-MOE 76 552467 3-9-4 2'-MOE 65 552523 4-9-3 2'-MOE 71 552468 3-9-4 2'-MOE 53 552524 4-9-3 2'-MOE 43 552469 3-9-4 2'-MOE 51 552525 4-9-3 2'-MOE 57 552470 3-9-4 2'-MOE 46 552526 4-9-3 2'-MOE 60 552471 3-9-4 2'-MOE 54 552527 4-9-3 2'-MOE 72 552472 3-9-4 2'-MOE 78 552528 4-9-3 2'-MOE 78 552473 3-9-4 2'-MOE 67 552529 4-9-3 2'-MOE 77 552474 3-9-4 2'-MOE 79 552530 4-9-3 2'-MOE 78 552475 3-9-4 2'-MOE 74 552531 4-9-3 2'-MOE 68 552476 3-9-4 2'-MOE 52 552477 3-9-4 2'-MOE 76 552478 3-9-4 2'-MOE 70 552479 3-9-4 2'-MOE 67 552480 3-9-4 2'-MOE 68 552481 3-9-4 2'-MOE 57 552482 3-9-4 2'-MOE 51 552483 3-9-4 2'-MOE 48 552484 3-9-4 2'-MOE 58 552485 3-9-4 2'-MOE 51 552486 3-9-4 2'-MOE 55 552487 3-9-4 2'-MOE 62 552488 3-9-4 2'-MOE 51 552489 3-9-4 2'-MOE 49 552490 3-9-4 2'-MOE 51 552491 3-9-4 2'-MOE 51 552492 3-9-4 2'-MOE 38 552493 3-9-4 2'-MOE 52 552494 3-9-4 2'-MOE 17 552495 3-9-4 2'-MOE 49

TABLE 82 Inhibition of viral Target-Z mRNA levels by chimeric antisense oligonucleotidesmeasured with RTS3370 Wing % ISIS No Motif chemistry inhibition 146786 5-10-5 2'-MOE 47 509959 3-10-3 2'-MOE 38 552552 5-9-2 2'-MOE 33 552553 5-9-2 2'-MOE 46 552554 5-9-2 2'-MOE 54 552555 5-9-2 2'-MOE 50 552556 5-9-2 2'-MOE 46 552557 5-9-2 2'-MOE 57 552558 5-9-2 2'-MOE 55 552559 5-9-2 2'-MOE 66 552560 5-9-2 2'-MOE 44 552561 5-9-2 2'-MOE 48 552562 5-9-2 2'-MOE 52 552563 5-9-2 2'-MOE 45 552564 5-9-2 2'-MOE 41 552565 5-9-2 2'-MOE 54 552566 5-9-2 2'-MOE 56 552567 5-9-2 2'-MOE 71 552568 5-9-2 2'-MOE 64 552569 5-9-2 2'-MOE 59 552570 5-9-2 2'-MOE 60 552571 5-9-2 2'-MOE 55 552572 5-9-2 2'-MOE 60 552573 5-9-2 2'-MOE 24 552574 5-9-2 2'-MOE 34 552575 5-9-2 2'-MOE 36 552576 5-9-2 2'-MOE 67 552577 5-9-2 2'-MOE 64 552578 5-9-2 2'-MOE 75 552579 5-9-2 2'-MOE 75 552580 5-9-2 2'-MOE 59 552581 5-9-2 2'-MOE 54 552582 5-9-2 2'-MOE 61 552583 5-9-2 2'-MOE 69 552584 5-9-2 2'-MOE 74 552585 5-9-2 2'-MOE 62 552586 5-9-2 2'-MOE 79 552587 5-9-2 2'-MOE 71 552532 4-9-3 2'-MOE 48 552588 5-9-2 2'-MOE 70 552533 4-9-3 2'-MOE 43 552589 5-9-2 2'-MOE 59 552534 4-9-3 2'-MOE 62 552590 5-9-2 2'-MOE 70 552535 4-9-3 2'-MOE 55 552591 5-9-2 2'-MOE 51 552536 4-9-3 2'-MOE 3 552592 5-9-2 2'-MOE 50 552537 4-9-3 2'-MOE 14 552593 5-9-2 2'-MOE 46 552538 4-9-3 2'-MOE 52 552594 5-9-2 2'-MOE 55 552539 4-9-3 2'-MOE 47 552595 5-9-2 2'-MOE 60 552540 4-9-3 2'-MOE 60 552596 5-9-2 2'-MOE 63 552541 4-9-3 2'-MOE 60 552597 5-9-2 2'-MOE 61 552542 4-9-3 2'-MOE 64 552598 5-9-2 2'-MOE 57 552543 4-9-3 2'-MOE 46 552600 5-9-2 2'-MOE 59 552544 4-9-3 2'-MOE 53 552602 5-9-2 2'-MOE 6 552545 4-9-3 2'-MOE 33 552604 5-9-2 2'-MOE 47 552546 4-9-3 2'-MOE 42 552606 5-9-2 2'-MOE 53 552547 4-9-3 2'-MOE 51 552608 5-9-2 2'-MOE 53 552548 4-9-3 2'-MOE 52 552610 5-9-2 2'-MOE 47 552549 4-9-3 2'-MOE 38 552612 5-9-2 2'-MOE 39 552550 4-9-3 2'-MOE 19 552614 5-9-2 2'-MOE 24 552551 4-9-3 2'-MOE 24 552616 5-9-2 2'-MOE 15

TABLE 83 Inhibition of viral Target-Z mRNA levels by chimeric antisense oligonucleotidesmeasured with RTS3370 Wing % ISIS No Motif chemistry inhibition 146786 5-10-5 2'-MOE 51 509934 5-10-5 2'-MOE 76 552007 6-10-4 2'-MOE 61 552039 7-10-3 2'-MOE 84 552008 6-10-4 2'-MOE 48 552040 7-10-3 2'-MOE 48 552009 6-10-4 2'-MOE 77 552041 7-10-3 2'-MOE 73 552010 6-10-4 2'-MOE 63 552042 7-10-3 2'-MOE 66 552011 6-10-4 2'-MOE 52 552043 7-10-3 2'-MOE 54 552012 6-10-4 2'-MOE 73 552044 7-10-3 2'-MOE 86 552013 6-10-4 2'-MOE 73 552045 7-10-3 2'-MOE 65 552014 6-10-4 2'-MOE 76 552046 7-10-3 2'-MOE 93 552015 6-10-4 2'-MOE 70 552047 7-10-3 2'-MOE 77 552016 6-10-4 2'-MOE 61 552048 7-10-3 2'-MOE 66 552017 6-10-4 2'-MOE 73 552049 7-10-3 2'-MOE 73 552018 6-10-4 2'-MOE 98 552050 7-10-3 2'-MOE 98 552019 6-10-4 2'-MOE 98 552051 7-10-3 2'-MOE 99 551986 4-10-6 2'-MOE 92 552020 6-10-4 2'-MOE 97 552052 7-10-3 2'-MOE 98 551987 4-10-6 2'-MOE 95 552021 6-10-4 2'-MOE 97 552053 7-10-3 2'-MOE 98 551988 4-10-6 2'-MOE 50 552005 5-10-5 2'-MOE 99 552022 6-10-4 2'-MOE 99 552054 7-10-3 2'-MOE 99 551989 4-10-6 2'-MOE 96 552023 6-10-4 2'-MOE 99 552055 7-10-3 2'-MOE 98 551990 4-10-6 2'-MOE 86 552024 6-10-4 2'-MOE 89 552056 7-10-3 2'-MOE 88 551991 4-10-6 2'-MOE 0 552025 6-10-4 2'-MOE 90 552057 7-10-3 2'-MOE 92 551992 4-10-6 2'-MOE 72 552026 6-10-4 2'-MOE 88 552058 7-10-3 2'-MOE 86 551993 4-10-6 2'-MOE 82 552027 6-10-4 2'-MOE 87 552059 7-10-3 2'-MOE 88 551994 4-10-6 2'-MOE 85 552028 6-10-4 2'-MOE 83 552060 7-10-3 2'-MOE 82 551995 4-10-6 2'-MOE 84 552029 6-10-4 2'-MOE 88 552061 7-10-3 2'-MOE 85 551996 4-10-6 2'-MOE 87 552030 6-10-4 2'-MOE 88 552062 7-10-3 2'-MOE 85 551997 4-10-6 2'-MOE 83 552031 6-10-4 2'-MOE 82 551998 4-10-6 2'-MOE 85 552032 6-10-4 2'-MOE 87 551999 4-10-6 2'-MOE 82 552033 6-10-4 2'-MOE 87 552000 4-10-6 2'-MOE 83 552006 5-10-5 2'-MOE 88 552034 6-10-4 2'-MOE 89 552001 4-10-6 2'-MOE 65 552035 6-10-4 2'-MOE 60 552002 4-10-6 2'-MOE 63 552036 6-10-4 2'-MOE 65 552003 4-10-6 2'-MOE 65 552037 6-10-4 2'-MOE 58 552004 4-10-6 2'-MOE 58 552038 6-10-4 2'-MOE 70

TABLE 84 Inhibition of viral Target-Z mRNA levels by chimeric antisense oligonucleotidesmeasured with RTS3370 Wing % ISIS No Motif chemistry inhibition 146786 5-10-5 2'-MOE 64 510100 3-10-4 2'-MOE 62 552168 3-9-5 2'-MOE 79 552222 4-9-4 2'-MOE 79 552169 3-9-5 2'-MOE 67 552223 4-9-4 2'-MOE 40 552170 3-9-5 2'-MOE 69 552224 4-9-4 2'-MOE 64 552171 3-9-5 2'-MOE 65 552225 4-9-4 2'-MOE 69 552172 3-9-5 2'-MOE 33 552226 4-9-4 2'-MOE 48 552173 3-9-5 2'-MOE 41 552227 4-9-4 2'-MOE 32 552174 3-9-5 2'-MOE 31 552228 4-9-4 2'-MOE 42 552175 3-9-5 2'-MOE 59 552176 3-9-5 2'-MOE 68 552177 3-9-5 2'-MOE 55 552178 3-9-5 2'-MOE 66 552179 3-9-5 2'-MOE 70 552180 3-9-5 2'-MOE 66 552181 3-9-5 2'-MOE 51 552182 3-9-5 2'-MOE 69 552183 3-9-5 2'-MOE 69 552184 3-9-5 2'-MOE 43 552185 3-9-5 2'-MOE 66 552186 3-9-5 2'-MOE 54 552187 3-9-5 2'-MOE 74 552188 3-9-5 2'-MOE 78 552189 3-9-5 2'-MOE 57 552190 3-9-5 2'-MOE 39 552191 3-9-5 2'-MOE 60 552192 3-9-5 2'-MOE 85 552193 3-9-5 2'-MOE 86 552194 3-9-5 2'-MOE 68 552195 3-9-5 2'-MOE 73 552196 3-9-5 2'-MOE 60 552197 3-9-5 2'-MOE 60 552198 3-9-5 2'-MOE 61 552199 3-9-5 2'-MOE 89 552200 3-9-5 2'-MOE 85 552201 3-9-5 2'-MOE 81 552202 3-9-5 2'-MOE 76 552203 3-9-5 2'-MOE 74 552204 3-9-5 2'-MOE 71 552151 2-9-6 2'-MOE 77 552205 3-9-5 2'-MOE 78 552152 2-9-6 2'-MOE 72 552206 3-9-5 2'-MOE 77 552153 2-9-6 2'-MOE 67 552207 3-9-5 2'-MOE 81 552154 2-9-6 2'-MOE 56 552208 3-9-5 2'-MOE 70 552155 2-9-6 2'-MOE 61 552209 3-9-5 2'-MOE 63 552156 2-9-6 2'-MOE 20 552210 3-9-5 2'-MOE 75 552157 2-9-6 2'-MOE 39 552211 3-9-5 2'-MOE 75 552158 2-9-6 2'-MOE 70 552212 3-9-5 2'-MOE 67 552159 2-9-6 2'-MOE 74 552213 3-9-5 2'-MOE 70 552160 2-9-6 2'-MOE 78 552214 3-9-5 2'-MOE 79 552161 2-9-6 2'-MOE 56 552215 3-9-5 2'-MOE 61 552162 2-9-6 2'-MOE 64 552216 3-9-5 2'-MOE 62 552163 2-9-6 2'-MOE 71 552217 3-9-5 2'-MOE 58 552164 2-9-6 2'-MOE 52 552218 3-9-5 2'-MOE 56 552165 2-9-6 2'-MOE 53 552219 3-9-5 2'-MOE 33 552166 2-9-6 2'-MOE 41 552220 3-9-5 2'-MOE 53 552167 2-9-6 2'-MOE 54 552221 3-9-5 2'-MOE 31

TABLE 85 Inhibition of viral Target-Z mRNA levels by chimeric antisense oligonucleotidesmeasured with RTS3370 Wing % ISIS No Motif chemistry inhibition 146786 5-10-5 2'-MOE 73 509934 5-10-5 2'-MOE 76 510100 3-10-4 2'-MOE 73 552071 8-10-2 2'-MOE 79 552114 2-9-6 2'-MOE 66 552115 2-9-6 2'-MOE 70 552116 2-9-6 2'-MOE 68 552117 2-9-6 2'-MOE 70 552072 8-10-2 2'-MOE 50 552118 2-9-6 2'-MOE 66 552119 2-9-6 2'-MOE 62 552120 2-9-6 2'-MOE 35 552121 2-9-6 2'-MOE 39 552073 8-10-2 2'-MOE 80 552122 2-9-6 2'-MOE 55 552074 8-10-2 2'-MOE 73 552123 2-9-6 2'-MOE 75 552075 8-10-2 2'-MOE 78 552124 2-9-6 2'-MOE 64 552076 8-10-2 2'-MOE 70 552125 2-9-6 2'-MOE 73 552077 8-10-2 2'-MOE 83 552126 2-9-6 2'-MOE 64 552078 8-10-2 2'-MOE 80 552127 2-9-6 2'-MOE 72 552079 8-10-2 2'-MOE 86 552128 2-9-6 2'-MOE 76 552080 8-10-2 2'-MOE 83 552129 2-9-6 2'-MOE 72 552131 2-9-6 2'-MOE 61 552132 2-9-6 2'-MOE 73 552133 2-9-6 2'-MOE 75 552081 8-10-2 2'-MOE 76 552134 2-9-6 2'-MOE 58 552135 2-9-6 2'-MOE 67 552136 2-9-6 2'-MOE 65 552137 2-9-6 2'-MOE 55 552082 8-10-2 2'-MOE 98 552138 2-9-6 2'-MOE 82 552083 8-10-2 2'-MOE 99 552139 2-9-6 2'-MOE 86 552084 8-10-2 2'-MOE 99 552140 2-9-6 2'-MOE 74 552085 8-10-2 2'-MOE 100 552141 2-9-6 2'-MOE 67 552086 8-10-2 2'-MOE 100 552142 2-9-6 2'-MOE 45 552087 8-10-2 2'-MOE 100 552143 2-9-6 2'-MOE 68 552144 2-9-6 2'-MOE 78 552145 2-9-6 2'-MOE 88 552146 2-9-6 2'-MOE 81 552088 8-10-2 2'-MOE 95 552147 2-9-6 2'-MOE 88 552089 8-10-2 2'-MOE 93 552148 2-9-6 2'-MOE 79 552090 8-10-2 2'-MOE 87 552149 2-9-6 2'-MOE 81 552091 8-10-2 2'-MOE 88 552092 8-10-2 2'-MOE 90 552093 8-10-2 2'-MOE 91 552094 8-10-2 2'-MOE 88 552063 7-10-3 2'-MOE 81 552095 8-10-2 2'-MOE 89 552064 7-10-3 2'-MOE 85 552096 8-10-2 2'-MOE 92 552065 7-10-3 2'-MOE 86 552097 8-10-2 2'-MOE 93 552066 7-10-3 2'-MOE 33 552098 8-10-2 2'-MOE 88 552067 7-10-3 2'-MOE 50 552099 8-10-2 2'-MOE 70 552068 7-10-3 2'-MOE 73 552100 8-10-2 2'-MOE 70 552069 7-10-3 2'-MOE 73 552101 8-10-2 2'-MOE 76 552070 7-10-3 2'-MOE 71 552102 8-10-2 2'-MOE 64

TABLE 86 Inhibition of viral Target-Z mRNA levels by chimeric antisense oligonucleotides measured with RTS3370 Wing % ISIS No Motif chemistry inhibition 146786 5-10-5 2′-MOE 84 510100 3-10-4 2′-MOE 76 552330 6-9-2 2′-MOE 54 552331 6-9-2 2′-MOE 66 552332 6-9-2 2′-MOE 70 552333 6-9-2 2′-MOE 55 552334 6-9-2 2′-MOE 42 552335 6-9-2 2′-MOE 39 552336 6-9-2 2′-MOE 27 552337 6-9-2 2′-MOE 74 552338 6-9-2 2′-MOE 68 552339 6-9-2 2′-MOE 71 552340 6-9-2 2′-MOE 61 552341 6-9-2 2′-MOE 58 552342 6-9-2 2′-MOE 55 552343 6-9-2 2′-MOE 63 552344 6-9-2 2′-MOE 51 552345 6-9-2 2′-MOE 65 552346 6-9-2 2′-MOE 0 552347 6-9-2 2′-MOE 84 552348 6-9-2 2′-MOE 87 552349 6-9-2 2′-MOE 74 552350 6-9-2 2′-MOE 59 552351 6-9-2 2′-MOE 60 552352 6-9-2 2′-MOE 53 552353 6-9-2 2′-MOE 0 552354 6-9-2 2′-MOE 83 552355 6-9-2 2′-MOE 90 552356 6-9-2 2′-MOE 0 552357 6-9-2 2′-MOE 45 552358 6-9-2 2′-MOE 74 552359 6-9-2 2′-MOE 72 552360 6-9-2 2′-MOE 87 552361 6-9-2 2′-MOE 96 552308 5-9-3 2′-MOE 81 552362 6-9-2 2′-MOE 92 552309 5-9-3 2′-MOE 77 552363 6-9-2 2′-MOE 92 552310 5-9-3 2′-MOE 80 552364 6-9-2 2′-MOE 87 552311 5-9-3 2′-MOE 13 552365 6-9-2 2′-MOE 84 552150 2-9-6 2′-MOE 73 552312 5-9-3 2′-MOE 77 552366 6-9-2 2′-MOE 87 552313 5-9-3 2′-MOE 64 552367 6-9-2 2′-MOE 85 552314 5-9-3 2′-MOE 73 552368 6-9-2 2′-MOE 77 552315 5-9-3 2′-MOE 75 552369 6-9-2 2′-MOE 75 552316 5-9-3 2′-MOE 64 552370 6-9-2 2′-MOE 63 552317 5-9-3 2′-MOE 99 552371 6-9-2 2′-MOE 81 552318 5-9-3 2′-MOE 76 552372 6-9-2 2′-MOE 65 552319 5-9-3 2′-MOE 55 552373 6-9-2 2′-MOE 74 552320 5-9-3 2′-MOE 68 552374 6-9-2 2′-MOE 78 552321 5-9-3 2′-MOE 74 552375 6-9-2 2′-MOE 81 552322 5-9-3 2′-MOE 73 552376 6-9-2 2′-MOE 78 552323 5-9-3 2′-MOE 75 552377 6-9-2 2′-MOE 70 552324 5-9-3 2′-MOE 0 552378 6-9-2 2′-MOE 72 552325 5-9-3 2′-MOE 70 552379 6-9-2 2′-MOE 74 552326 5-9-3 2′-MOE 63 552380 6-9-2 2′-MOE 53 552327 5-9-3 2′-MOE 30 552381 6-9-2 2′-MOE 26 552328 5-9-3 2′-MOE 25 552382 6-9-2 2′-MOE 13 552329 5-9-3 2′-MOE 33 552383 6-9-2 2′-MOE 5

TABLE 87 Inhibition of viral Target-Z mRNA levels by chimeric antisense oligonucleotides measured with RTS3370 Wing % ISIS No Motif chemistry inhibition 509934 5-10-5 2′-MOE 30 551909 2-10-8 2′-MOE 62 551941 3-10-7 2′-MOE 74 551973 4-10-6 2′-MOE 64 551910 2-10-8 2′-MOE 52 551942 3-10-7 2′-MOE 54 551974 4-10-6 2′-MOE 51 551911 2-10-8 2′-MOE 58 551943 3-10-7 2′-MOE 64 551975 4-10-6 2′-MOE 57 551912 2-10-8 2′-MOE 59 551944 3-10-7 2′-MOE 66 551976 4-10-6 2′-MOE 57 551913 2-10-8 2′-MOE 58 551945 3-10-7 2′-MOE 56 551977 4-10-6 2′-MOE 56 551914 2-10-8 2′-MOE 0 551946 3-10-7 2′-MOE 48 551978 4-10-6 2′-MOE 53 551915 2-10-8 2′-MOE 44 551947 3-10-7 2′-MOE 53 551979 4-10-6 2′-MOE 64 551916 2-10-8 2′-MOE 57 551948 3-10-7 2′-MOE 68 551980 4-10-6 2′-MOE 56 551917 2-10-8 2′-MOE 58 551949 3-10-7 2′-MOE 64 551981 4-10-6 2′-MOE 63 551918 2-10-8 2′-MOE 59 551950 3-10-7 2′-MOE 71 551982 4-10-6 2′-MOE 63 551919 2-10-8 2′-MOE 76 551951 3-10-7 2′-MOE 71 551983 4-10-6 2′-MOE 73 551920 2-10-8 2′-MOE 68 551952 3-10-7 2′-MOE 76 551984 4-10-6 2′-MOE 81 551921 2-10-8 2′-MOE 83 551953 3-10-7 2′-MOE 82 551985 4-10-6 2′-MOE 76 551922 2-10-8 2′-MOE 73 551954 3-10-7 2′-MOE 68 551923 2-10-8 2′-MOE 59 551955 3-10-7 2′-MOE 71 551924 2-10-8 2′-MOE 80 551956 3-10-7 2′-MOE 80 551925 2-10-8 2′-MOE 82 551957 3-10-7 2′-MOE 88 551926 2-10-8 2′-MOE 71 551958 3-10-7 2′-MOE 74 551927 2-10-8 2′-MOE 68 551959 3-10-7 2′-MOE 69 551928 2-10-8 2′-MOE 69 551960 3-10-7 2′-MOE 62 551929 2-10-8 2′-MOE 54 551961 3-10-7 2′-MOE 20 551930 2-10-8 2′-MOE 53 551962 3-10-7 2′-MOE 60 551931 2-10-8 2′-MOE 47 551963 3-10-7 2′-MOE 63 551932 2-10-8 2′-MOE 68 551964 3-10-7 2′-MOE 56 551933 2-10-8 2′-MOE 72 551965 3-10-7 2′-MOE 67 551934 2-10-8 2′-MOE 64 551966 3-10-7 2′-MOE 73 551935 2-10-8 2′-MOE 68 551967 3-10-7 2′-MOE 60 551936 2-10-8 2′-MOE 67 551968 3-10-7 2′-MOE 63 551937 2-10-8 2′-MOE 47 551969 3-10-7 2′-MOE 36 551938 2-10-8 2′-MOE 41 551970 3-10-7 2′-MOE 43 551939 2-10-8 2′-MOE 53 551971 3-10-7 2′-MOE 55 551940 2-10-8 2′-MOE 50 551972 3-10-7 2′-MOE 58

TABLE 88 Inhibition of viral Target-Z mRNA levels by chimeric antisense oligonucleotides measured with RTS3371 Wing % ISIS No Motif chemistry inhibition 509934 5-10-5 2′-MOE 21 551909 2-10-8 2′-MOE 52 551941 3-10-7 2′-MOE 62 551973 4-10-6 2′-MOE 58 551910 2-10-8 2′-MOE 48 551942 3-10-7 2′-MOE 36 551974 4-10-6 2′-MOE 45 551911 2-10-8 2′-MOE 61 551943 3-10-7 2′-MOE 56 551975 4-10-6 2′-MOE 60 551912 2-10-8 2′-MOE 53 551944 3-10-7 2′-MOE 48 551976 4-10-6 2′-MOE 48 551913 2-10-8 2′-MOE 53 551945 3-10-7 2′-MOE 54 551977 4-10-6 2′-MOE 48 551914 2-10-8 2′-MOE 0 551946 3-10-7 2′-MOE 56 551978 4-10-6 2′-MOE 36 551915 2-10-8 2′-MOE 47 551947 3-10-7 2′-MOE 45 551979 4-10-6 2′-MOE 54 551916 2-10-8 2′-MOE 44 551948 3-10-7 2′-MOE 59 551980 4-10-6 2′-MOE 49 551917 2-10-8 2′-MOE 48 551949 3-10-7 2′-MOE 60 551981 4-10-6 2′-MOE 57 551918 2-10-8 2′-MOE 53 551950 3-10-7 2′-MOE 57 551982 4-10-6 2′-MOE 57 551919 2-10-8 2′-MOE 65 551951 3-10-7 2′-MOE 57 551983 4-10-6 2′-MOE 53 551920 2-10-8 2′-MOE 57 551952 3-10-7 2′-MOE 67 551984 4-10-6 2′-MOE 62 551921 2-10-8 2′-MOE 60 551953 3-10-7 2′-MOE 57 551985 4-10-6 2′-MOE 58 551922 2-10-8 2′-MOE 63 551954 3-10-7 2′-MOE 61 551923 2-10-8 2′-MOE 50 551955 3-10-7 2′-MOE 44 551924 2-10-8 2′-MOE 52 551956 3-10-7 2′-MOE 46 551925 2-10-8 2′-MOE 54 551957 3-10-7 2′-MOE 51 551926 2-10-8 2′-MOE 70 551958 3-10-7 2′-MOE 72 551927 2-10-8 2′-MOE 60 551959 3-10-7 2′-MOE 61 551928 2-10-8 2′-MOE 57 551960 3-10-7 2′-MOE 58 551929 2-10-8 2′-MOE 49 551961 3-10-7 2′-MOE 26 551930 2-10-8 2′-MOE 54 551962 3-10-7 2′-MOE 57 551931 2-10-8 2′-MOE 46 551963 3-10-7 2′-MOE 56 551932 2-10-8 2′-MOE 57 551964 3-10-7 2′-MOE 53 551933 2-10-8 2′-MOE 65 551965 3-10-7 2′-MOE 54 551934 2-10-8 2′-MOE 58 551966 3-10-7 2′-MOE 69 551935 2-10-8 2′-MOE 63 551967 3-10-7 2′-MOE 53 551936 2-10-8 2′-MOE 67 551968 3-10-7 2′-MOE 60 551937 2-10-8 2′-MOE 51 551969 3-10-7 2′-MOE 42 551938 2-10-8 2′-MOE 40 551970 3-10-7 2′-MOE 38 551939 2-10-8 2′-MOE 32 551971 3-10-7 2′-MOE 46 551940 2-10-8 2′-MOE 39 551972 3-10-7 2′-MOE 51

TABLE 89 Inhibition of viral Target-Z mRNA levels by chimeric antisense oligonucleotides measured with RTS3371 Wing % ISIS No Motif chemistry inhibition 146786 5-10-5 2′-MOE 40 510100 3-10-4 2′-MOE 60 552276 5-9-3 2′-MOE 44 552277 5-9-3 2′-MOE 39 552278 5-9-3 2′-MOE 37 552279 5-9-3 2′-MOE 50 552280 5-9-3 2′-MOE 2 552281 5-9-3 2′-MOE 0 552282 5-9-3 2′-MOE 13 552229 4-9-4 2′-MOE 17 552283 5-9-3 2′-MOE 27 552230 4-9-4 2′-MOE 53 552284 5-9-3 2′-MOE 0 552231 4-9-4 2′-MOE 31 552285 5-9-3 2′-MOE 56 552232 4-9-4 2′-MOE 35 552286 5-9-3 2′-MOE 43 552233 4-9-4 2′-MOE 40 552287 5-9-3 2′-MOE 44 552234 4-9-4 2′-MOE 0 552288 5-9-3 2′-MOE 44 552235 4-9-4 2′-MOE 13 552289 5-9-3 2′-MOE 21 552236 4-9-4 2′-MOE 40 552290 5-9-3 2′-MOE 34 552237 4-9-4 2′-MOE 37 552291 5-9-3 2′-MOE 34 552239 4-9-4 2′-MOE 58 552293 5-9-3 2′-MOE 61 552240 4-9-4 2′-MOE 54 552294 5-9-3 2′-MOE 62 552241 4-9-4 2′-MOE 47 552295 5-9-3 2′-MOE 63 552242 4-9-4 2′-MOE 61 552296 5-9-3 2′-MOE 61 552243 4-9-4 2′-MOE 55 552297 5-9-3 2′-MOE 52 552244 4-9-4 2′-MOE 45 552298 5-9-3 2′-MOE 27 552245 4-9-4 2′-MOE 41 552299 5-9-3 2′-MOE 32 552246 4-9-4 2′-MOE 67 552300 5-9-3 2′-MOE 57 552247 4-9-4 2′-MOE 74 552301 5-9-3 2′-MOE 76 552248 4-9-4 2′-MOE 65 552302 5-9-3 2′-MOE 68 552249 4-9-4 2′-MOE 38 552303 5-9-3 2′-MOE 59 552250 4-9-4 2′-MOE 43 552304 5-9-3 2′-MOE 30 552251 4-9-4 2′-MOE 52 552305 5-9-3 2′-MOE 49 552252 4-9-4 2′-MOE 51 552306 5-9-3 2′-MOE 56 552253 4-9-4 2′-MOE 47 552307 5-9-3 2′-MOE 49 552254 4-9-4 2′-MOE 50 552255 4-9-4 2′-MOE 64 552256 4-9-4 2′-MOE 57 552257 4-9-4 2′-MOE 51 552258 4-9-4 2′-MOE 62 552259 4-9-4 2′-MOE 59 552260 4-9-4 2′-MOE 56 552261 4-9-4 2′-MOE 54 552262 4-9-4 2′-MOE 47 552263 4-9-4 2′-MOE 45 552264 4-9-4 2′-MOE 52 552265 4-9-4 2′-MOE 58 552266 4-9-4 2′-MOE 54 552267 4-9-4 2′-MOE 43 552268 4-9-4 2′-MOE 57 552269 4-9-4 2′-MOE 34 552270 4-9-4 2′-MOE 37 552271 4-9-4 2′-MOE 42 552272 4-9-4 2′-MOE 36 552273 4-9-4 2′-MOE 25 552274 4-9-4 2′-MOE 11 552275 4-9-4 2′-MOE 38

TABLE 90 Inhibition of viral Target-Z mRNA levels by chimeric antisense oligonucleotides measured with RTS3371 Wing % ISIS No Motif chemistry inhibition 146786 5-10-5 2′-MOE 38 509959 3-10-3 2′-MOE 49 510100 3-10-4 2′-MOE 55 552384 2-9-5 2′-MOE 41 552440 3-9-4 2′-MOE 57 552385 2-9-5 2′-MOE 53 552441 3-9-4 2′-MOE 38 552386 2-9-5 2′-MOE 42 552442 3-9-4 2′-MOE 72 552387 2-9-5 2′-MOE 43 552443 3-9-4 2′-MOE 56 552388 2-9-5 2′-MOE 18 552444 3-9-4 2′-MOE 39 552389 2-9-5 2′-MOE 24 552445 3-9-4 2′-MOE 53 552390 2-9-5 2′-MOE 40 552446 3-9-4 2′-MOE 57 552391 2-9-5 2′-MOE 51 552447 3-9-4 2′-MOE 53 552392 2-9-5 2′-MOE 0 552448 3-9-4 2′-MOE 57 552393 2-9-5 2′-MOE 52 552449 3-9-4 2′-MOE 49 552394 2-9-5 2′-MOE 32 552450 3-9-4 2′-MOE 44 552395 2-9-5 2′-MOE 33 552451 3-9-4 2′-MOE 38 552396 2-9-5 2′-MOE 46 552452 3-9-4 2′-MOE 30 552130 2-9-6 2′-MOE 46 552184 3-9-5 2′-MOE 34 552238 4-9-4 2′-MOE 41 552292 5-9-3 2′-MOE 45 552346 6-9-2 2′-MOE 0 552397 2-9-5 2′-MOE 37 552453 3-9-4 2′-MOE 45 552398 2-9-5 2′-MOE 42 552454 3-9-4 2′-MOE 39 552399 2-9-5 2′-MOE 34 552400 2-9-5 2′-MOE 47 552401 2-9-5 2′-MOE 53 552402 2-9-5 2′-MOE 47 552403 2-9-5 2′-MOE 70 552404 2-9-5 2′-MOE 44 552405 2-9-5 2′-MOE 0 552406 2-9-5 2′-MOE 25 552407 2-9-5 2′-MOE 23 552408 2-9-5 2′-MOE 73 552409 2-9-5 2′-MOE 71 552410 2-9-5 2′-MOE 52 552411 2-9-5 2′-MOE 62 552412 2-9-5 2′-MOE 50 552413 2-9-5 2′-MOE 55 552414 2-9-5 2′-MOE 64 552415 2-9-5 2′-MOE 45 552416 2-9-5 2′-MOE 45 552417 2-9-5 2′-MOE 37 552418 2-9-5 2′-MOE 73 552419 2-9-5 2′-MOE 68 552420 2-9-5 2′-MOE 64 552421 2-9-5 2′-MOE 54 552422 2-9-5 2′-MOE 60 552423 2-9-5 2′-MOE 62 552424 2-9-5 2′-MOE 60 552425 2-9-5 2′-MOE 46 552426 2-9-5 2′-MOE 48 552427 2-9-5 2′-MOE 36 552428 2-9-5 2′-MOE 57 552429 2-9-5 2′-MOE 36 552430 2-9-5 2′-MOE 42 552431 2-9-5 2′-MOE 60 552432 2-9-5 2′-MOE 44 552433 2-9-5 2′-MOE 55 552434 2-9-5 2′-MOE 46 552435 2-9-5 2′-MOE 47 552436 2-9-5 2′-MOE 25 552437 2-9-5 2′-MOE 19 552438 2-9-5 2′-MOE 25 552439 2-9-5 2′-MOE 22

TABLE 91 Inhibition of viral Target-Z mRNA levels by chimeric antisense oligonucleotides measured with RTS3371 Wing % ISIS No Motif chemistry inhibition 509959 3-10-3 2′-MOE 49 552496 4-9-3 2′-MOE 35 552497 4-9-3 2′-MOE 60 552498 4-9-3 2′-MOE 20 552499 4-9-3 2′-MOE 45 552500 4-9-3 2′-MOE 53 552501 4-9-3 2′-MOE 56 552502 4-9-3 2′-MOE 50 552503 4-9-3 2′-MOE 36 552504 4-9-3 2′-MOE 50 552505 4-9-3 2′-MOE 53 552506 4-9-3 2′-MOE 49 552507 4-9-3 2′-MOE 35 552508 4-9-3 2′-MOE 62 552509 4-9-3 2′-MOE 65 552510 4-9-3 2′-MOE 54 552455 3-9-4 2′-MOE 60 552511 4-9-3 2′-MOE 65 552456 3-9-4 2′-MOE 69 552512 4-9-3 2′-MOE 63 552457 3-9-4 2′-MOE 4 552513 4-9-3 2′-MOE 50 552458 3-9-4 2′-MOE 59 552514 4-9-3 2′-MOE 53 552459 3-9-4 2′-MOE 69 552515 4-9-3 2′-MOE 68 552460 3-9-4 2′-MOE 3 552516 4-9-3 2′-MOE 65 552461 3-9-4 2′-MOE 37 552517 4-9-3 2′-MOE 54 552462 3-9-4 2′-MOE 42 552518 4-9-3 2′-MOE 23 552463 3-94 2′-MOE 28 552519 4-9-3 2′-MOE 32 552464 3-9-4 2′-MOE 72 552520 4-9-3 2′-MOE 61 552465 3-9-4 2′-MOE 68 552521 4-9-3 2′-MOE 68 552466 3-9-4 2′-MOE 76 552522 4-9-3 2′-MOE 71 552467 3-9-4 2′-MOE 72 552523 4-9-3 2′-MOE 73 552468 3-9-4 2′-MOE 50 552524 4-9-3 2′-MOE 49 552469 3-9-4 2′-MOE 65 552525 4-9-3 2′-MOE 45 552470 3-9-4 2′-MOE 58 552526 4-9-3 2′-MOE 39 552471 3-9-4 2′-MOE 30 552527 4-9-3 2′-MOE 39 552472 3-9-4 2′-MOE 43 552528 4-9-3 2′-MOE 43 552473 3-9-4 2′-MOE 25 552529 4-9-3 2′-MOE 50 552474 3-9-4 2′-MOE 70 552530 4-9-3 2′-MOE 73 552475 3-9-4 2′-MOE 64 552531 4-9-3 2′-MOE 62 552476 3-9-4 2′-MOE 50 552477 3-9-4 2′-MOE 66 552478 3-9-4 2′-MOE 68 552479 3-9-4 2′-MOE 60 552480 3-9-4 2′-MOE 58 552481 3-9-4 2′-MOE 54 552482 3-9-4 2′-MOE 44 552483 3-9-4 2′-MOE 17 552484 3-9-4 2′-MOE 64 552485 3-9-4 2′-MOE 56 552486 3-9-4 2′-MOE 26 552487 3-9-4 2′-MOE 42 552488 3-9-4 2′-MOE 35 552489 3-9-4 2′-MOE 46 552490 3-9-4 2′-MOE 41 552491 3-9-4 2′-MOE 38 552492 3-9-4 2′-MOE 47 552493 3-9-4 2′-MOE 49 552494 3-9-4 2′-MOE 22 552495 3-9-4 2′-MOE 0

TABLE 92 Inhibition of viral Target-Z mRNA levels by chimeric antisense oligonucleotides measured with RTS3371 Wing % ISIS No Motif chemistry inhibition 146786 5-10-5 2′-MOE 56 509959 3-10-3 2′-MOE 54 552552 5-9-2 2′-MOE 32 552553 5-9-2 2′-MOE 53 552554 5-9-2 2′-MOE 48 552555 5-9-2 2′-MOE 39 552556 5-9-2 2′-MOE 39 552557 5-9-2 2′-MOE 54 552558 5-9-2 2′-MOE 41 552559 5-9-2 2′-MOE 56 552560 5-9-2 2′-MOE 39 552561 5-9-2 2′-MOE 51 552562 5-9-2 2′-MOE 56 552563 5-9-2 2′-MOE 31 552564 5-9-2 2′-MOE 31 552565 5-9-2 2′-MOE 53 552566 5-9-2 2′-MOE 46 552567 5-9-2 2′-MOE 63 552568 5-9-2 2′-MOE 66 552569 5-9-2 2′-MOE 60 552570 5-9-2 2′-MOE 60 552571 5-9-2 2′-MOE 44 552572 5-9-2 2′-MOE 52 552573 5-9-2 2′-MOE 20 552574 5-9-2 2′-MOE 36 552575 5-9-2 2′-MOE 19 552576 5-9-2 2′-MOE 61 552577 5-9-2 2′-MOE 57 552578 5-9-2 2′-MOE 71 552579 5-9-2 2′-MOE 59 552580 5-9-2 2′-MOE 58 552581 5-9-2 2′-MOE 51 552582 5-9-2 2′-MOE 40 552583 5-9-2 2′-MOE 35 552584 5-9-2 2′-MOE 50 552585 5-9-2 2′-MOE 48 552586 5-9-2 2′-MOE 74 552587 5-9-2 2′-MOE 68 552532 4-9-3 2′-MOE 59 552588 5-9-2 2′-MOE 67 552533 4-9-3 2′-MOE 52 552589 5-9-2 2′-MOE 47 552534 4-9-3 2′-MOE 71 552590 5-9-2 2′-MOE 58 552535 4-9-3 2′-MOE 59 552591 5-9-2 2′-MOE 46 552536 4-9-3 2′-MOE 19 552592 5-9-2 2′-MOE 44 552537 4-9-3 2′-MOE 26 552593 5-9-2 2′-MOE 39 552538 4-9-3 2′-MOE 54 552594 5-9-2 2′-MOE 52 552539 4-9-3 2′-MOE 50 552595 5-9-2 2′-MOE 57 552540 4-9-3 2′-MOE 60 552596 5-9-2 2′-MOE 58 552541 4-9-3 2′-MOE 68 552597 5-9-2 2′-MOE 52 552542 4-9-3 2′-MOE 63 552598 5-9-2 2′-MOE 51 552543 4-9-3 2′-MOE 44 552600 5-9-2 2′-MOE 51 552544 4-9-3 2′-MOE 45 552602 5-9-2 2′-MOE 13 552545 4-9-3 2′-MOE 42 552604 5-9-2 2′-MOE 42 552546 4-9-3 2′-MOE 46 552606 5-9-2 2′-MOE 42 552547 4-9-3 2′-MOE 38 552608 5-9-2 2′-MOE 37 552548 4-9-3 2′-MOE 49 552610 5-9-2 2′-MOE 41 552549 4-9-3 2′-MOE 34 552612 5-9-2 2′-MOE 23 552550 4-9-3 2′-MOE 13 552614 5-9-2 2′-MOE 11 552551 4-9-3 2′-MOE 8 552616 5-9-2 2′-MOE 6

TABLE 93 Inhibition of viral Target-Z mRNA levels by chimeric antisense oligonucleotides measured with RTS3371 Wing % ISIS No Motif chemistry inhibition 146786 5-10-5 2′-MOE 47 509934 5-10-5 2′-MOE 67 552007 6-10-4 2′-MOE 53 552039 7-10-3 2′-MOE 74 552008 6-10-4 2′-MOE 47 552040 7-10-3 2′-MOE 57 552009 6-10-4 2′-MOE 70 552041 7-10-3 2′-MOE 65 552010 6-10-4 2′-MOE 51 552042 7-10-3 2′-MOE 59 552011 6-10-4 2′-MOE 47 552043 7-10-3 2′-MOE 36 552012 6-10-4 2′-MOE 62 552044 7-10-3 2′-MOE 82 552013 6-10-4 2′-MOE 72 552045 7-10-3 2′-MOE 62 552014 6-10-4 2′-MOE 73 552046 7-10-3 2′-MOE 74 552015 6-10-4 2′-MOE 66 552047 7-10-3 2′-MOE 60 552016 6-10-4 2′-MOE 67 552048 7-10-3 2′-MOE 60 552017 6-10-4 2′-MOE 72 552049 7-10-3 2′-MOE 68 552018 6-10-4 2′-MOE 89 552050 7-10-3 2′-MOE 86 552019 6-10-4 2′-MOE 87 552051 7-10-3 2′-MOE 86 551986 4-10-6 2′-MOE 64 552020 6-10-4 2′-MOE 86 552052 7-10-3 2′-MOE 87 551987 4-10-6 2′-MOE 76 552021 6-10-4 2′-MOE 84 552053 7-10-3 2′-MOE 75 551988 4-10-6 2′-MOE 5 552005 5-10-5 2′-MOE 72 552022 6-10-4 2′-MOE 80 552054 7-10-3 2′-MOE 83 551989 4-10-6 2′-MOE 64 552023 6-10-4 2′-MOE 78 552055 7-10-3 2′-MOE 57 551990 4-10-6 2′-MOE 83 552024 6-10-4 2′-MOE 89 552056 7-10-3 2′-MOE 82 551991 4-10-6 2′-MOE 0 552025 6-10-4 2′-MOE 89 552057 7-10-3 2′-MOE 89 551992 4-10-6 2′-MOE 67 552026 6-10-4 2′-MOE 84 552058 7-10-3 2′-MOE 82 551993 4-10-6 2′-MOE 78 552027 6-10-4 2′-MOE 85 552059 7-10-3 2′-MOE 85 551994 4-10-6 2′-MOE 82 552028 6-10-4 2′-MOE 82 552060 7-10-3 2′-MOE 74 551995 4-10-6 2′-MOE 81 552029 6-10-4 2′-MOE 81 552061 7-10-3 2′-MOE 81 551996 4-10-6 2′-MOE 79 552030 6-10-4 2′-MOE 86 552062 7-10-3 2′-MOE 85 551997 4-10-6 2′-MOE 80 552031 6-10-4 2′-MOE 86 551998 4-10-6 2′-MOE 74 552032 6-10-4 2′-MOE 78 551999 4-10-6 2′-MOE 79 552033 6-10-4 2′-MOE 80 552000 4-10-6 2′-MOE 84 552006 5-10-5 2′-MOE 86 552034 6-10-4 2′-MOE 81 552001 4-10-6 2′-MOE 66 552035 6-10-4 2′-MOE 55 552002 4-10-6 2′-MOE 54 552036 6-10-4 2′-MOE 58 552003 4-10-6 2′-MOE 50 552037 6-10-4 2′-MOE 43 552004 4-10-6 2′-MOE 56 552038 6-10-4 2′-MOE 66

TABLE 94 Inhibition of viral Target-Z mRNA levels by chimeric antisense oligonucleotides measured with RTS3371 Wing % ISIS No Motif chemistry inhibition 146786 5-10-5 2′-MOE 61 510100 3-10-4 2′-MOE 66 552168 3-9-5 2′-MOE 64 552222 4-9-4 2′-MOE 76 552169 3-9-5 2′-MOE 65 552223 4-9-4 2′-MOE 41 552170 3-9-5 2′-MOE 58 552224 4-9-4 2′-MOE 58 552171 3-9-5 2′-MOE 51 552225 4-9-4 2′-MOE 49 552172 3-9-5 2′-MOE 23 552226 4-9-4 2′-MOE 36 552173 3-9-5 2′-MOE 44 552227 4-9-4 2′-MOE 20 552174 3-9-5 2′-MOE 28 552228 4-9-4 2′-MOE 29 552175 3-9-5 2′-MOE 56 552176 3-9-5 2′-MOE 66 552177 3-9-5 2′-MOE 53 552178 3-9-5 2′-MOE 57 552179 3-9-5 2′-MOE 56 552180 3-9-5 2′-MOE 51 552181 3-9-5 2′-MOE 51 552182 3-9-5 2′-MOE 63 552183 3-9-5 2′-MOE 60 552185 3-9-5 2′-MOE 67 552186 3-9-5 2′-MOE 37 552187 3-9-5 2′-MOE 68 552188 3-9-5 2′-MOE 71 552189 3-9-5 2′-MOE 51 552190 3-9-5 2′-MOE 47 552191 3-9-5 2′-MOE 50 552192 3-9-5 2′-MOE 80 552193 3-9-5 2′-MOE 73 552194 3-9-5 2′-MOE 58 552195 3-9-5 2′-MOE 60 552196 3-9-5 2′-MOE 54 552197 3-9-5 2′-MOE 64 552198 3-9-5 2′-MOE 62 552199 3-9-5 2′-MOE 57 552200 3-9-5 2′-MOE 52 552201 3-9-5 2′-MOE 73 552202 3-9-5 2′-MOE 60 552203 3-9-5 2′-MOE 60 552204 3-9-5 2′-MOE 63 552151 2-9-6 2′-MOE 71 552205 3-9-5 2′-MOE 64 552152 2-9-6 2′-MOE 69 552206 3-9-5 2′-MOE 71 552153 2-9-6 2′-MOE 63 552207 3-9-5 2′-MOE 71 552154 2-9-6 2′-MOE 56 552208 3-9-5 2′-MOE 52 552155 2-9-6 2′-MOE 61 552209 3-9-5 2′-MOE 50 552156 2-9-6 2′-MOE 40 552210 3-9-5 2′-MOE 66 552157 2-9-6 2′-MOE 45 552211 3-9-5 2′-MOE 63 552158 2-9-6 2′-MOE 66 552212 3-9-5 2′-MOE 62 552159 2-9-6 2′-MOE 68 552213 3-9-5 2′-MOE 64 552160 2-9-6 2′-MOE 78 552214 3-9-5 2′-MOE 72 552161 2-9-6 2′-MOE 57 552215 3-9-5 2′-MOE 54 552162 2-9-6 2′-MOE 54 552216 3-9-5 2′-MOE 49 552163 2-9-6 2′-MOE 65 552217 3-9-5 2′-MOE 50 552164 2-9-6 2′-MOE 48 552218 3-9-5 2′-MOE 39 552165 2-9-6 2′-MOE 46 552219 3-9-5 2′-MOE 41 552166 2-9-6 2′-MOE 42 552220 3-9-5 2′-MOE 32 552167 2-9-6 2′-MOE 47 552221 3-9-5 2′-MOE 33

TABLE 95 Inhibition of viral Target-Z mRNA levels by chimeric antisense oligonucleotides measured with RTS3371 Wing % ISIS No Motif chemistry inhibition 146786 5-10-5 2′-MOE 509934 5-10-5 2′-MOE 56 510100 3-10-4 2′-MOE 69 552071 8-10-2 2′-MOE 73 552114 2-9-6 2′-MOE 64 552115 2-9-6 2′-MOE 61 552116 2-9-6 2′-MOE 53 552117 2-9-6 2′-MOE 69 552072 8-10-2 2′-MOE 39 552118 2-9-6 2′-MOE 49 552119 2-9-6 2′-MOE 49 552120 2-9-6 2′-MOE 21 552121 2-9-6 2′-MOE 27 552073 8-10-2 2′-MOE 73 552122 2-9-6 2′-MOE 48 552074 8-10-2 2′-MOE 69 552123 2-9-6 2′-MOE 68 552075 8-10-2 2′-MOE 78 552124 2-9-6 2′-MOE 47 552076 8-10-2 2′-MOE 63 552125 2-9-6 2′-MOE 72 552077 8-10-2 2′-MOE 62 552126 2-9-6 2′-MOE 64 552078 8-10-2 2′-MOE 59 552127 2-9-6 2′-MOE 65 552079 8-10-2 2′-MOE 80 552128 2-9-6 2′-MOE 78 552080 8-10-2 2′-MOE 74 552129 2-9-6 2′-MOE 68 552130 2-9-6 2′-MOE 46 552131 2-9-6 2′-MOE 61 552132 2-9-6 2′-MOE 66 552133 2-9-6 2′-MOE 78 552081 8-10-2 2′-MOE 69 552134 2-9-6 2′-MOE 68 552135 2-9-6 2′-MOE 59 552136 2-9-6 2′-MOE 39 552137 2-9-6 2′-MOE 36 552082 8-10-2 2′-MOE 86 552138 2-9-6 2′-MOE 80 552083 8-10-2 2′-MOE 85 552139 2-9-6 2′-MOE 80 552084 8-10-2 2′-MOE 86 552140 2-9-6 2′-MOE 70 552085 8-10-2 2′-MOE 83 552141 2-9-6 2′-MOE 72 552086 8-10-2 2′-MOE 83 552142 2-9-6 2′-MOE 58 552087 8-10-2 2′-MOE 77 552143 2-9-6 2′-MOE 70 552144 2-9-6 2′-MOE 66 552145 2-9-6 2′-MOE 78 552146 2-9-6 2′-MOE 63 552088 8-10-2 2′-MOE 90 552147 2-9-6 2′-MOE 80 552089 8-10-2 2′-MOE 87 552148 2-9-6 2′-MOE 74 552090 8-10-2 2′-MOE 85 552149 2-9-6 2′-MOE 79 552091 8-10-2 2′-MOE 84 552092 8-10-2 2′-MOE 86 552093 8-10-2 2′-MOE 82 552094 8-10-2 2′-MOE 84 552063 7-10-3 2′-MOE 79 552095 8-10-2 2′-MOE 85 552064 7-10-3 2′-MOE 83 552096 8-10-2 2′-MOE 88 552065 7-10-3 2′-MOE 86 552097 8-10-2 2′-MOE 90 552066 7-10-3 2′-MOE 35 552098 8-10-2 2′-MOE 86 552067 7-10-3 2′-MOE 53 552099 8-10-2 2′-MOE 66 552068 7-10-3 2′-MOE 70 552100 8-10-2 2′-MOE 67 552069 7-10-3 2′-MOE 68 552101 8-10-2 2′-MOE 65 552070 7-10-3 2′-MOE 64 552102 8-10-2 2′-MOE 54

TABLE 96 Inhibition of viral Target-Z mRNA levels by chimeric antisense oligonucleotides measured with RTS3371 Wing % ISIS No Motif chemistry inhibition 146786 5-10-5 2′-MOE 63 510100 3-10-4 2′-MOE 59 552330 6-9-2 2′-MOE 50 552331 6-9-2 2′-MOE 46 552332 6-9-2 2′-MOE 50 552333 6-9-2 2′-MOE 48 552334 6-9-2 2′-MOE 42 552335 6-9-2 2′-MOE 30 552336 6-9-2 2′-MOE 23 552337 6-9-2 2′-MOE 42 552338 6-9-2 2′-MOE 40 552339 6-9-2 2′-MOE 50 552340 6-9-2 2′-MOE 45 552341 6-9-2 2′-MOE 44 552342 6-9-2 2′-MOE 51 552343 6-9-2 2′-MOE 44 552344 6-9-2 2′-MOE 24 552345 6-9-2 2′-MOE 41 552346 6-9-2 2′-MOE 0 552347 6-9-2 2′-MOE 75 552348 6-9-2 2′-MOE 72 552349 6-9-2 2′-MOE 65 552350 6-9-2 2′-MOE 42 552351 6-9-2 2′-MOE 45 552352 6-9-2 2′-MOE 43 552353 6-9-2 2′-MOE 20 552354 6-9-2 2′-MOE 70 552355 6-9-2 2′-MOE 66 552356 6-9-2 2′-MOE 62 552357 6-9-2 2′-MOE 53 552358 6-9-2 2′-MOE 57 552359 6-9-2 2′-MOE 46 552360 6-9-2 2′-MOE 45 552361 6-9-2 2′-MOE 44 552308 5-9-3 2′-MOE 38 552362 6-9-2 2′-MOE 51 552309 5-9-3 2′-MOE 76 552363 6-9-2 2′-MOE 73 552310 5-9-3 2′-MOE 58 552364 6-9-2 2′-MOE 66 552311 5-9-3 2′-MOE 38 552365 6-9-2 2′-MOE 64 552150 2-9-6 2′-MOE 68 552312 5-9-3 2′-MOE 75 552366 6-9-2 2′-MOE 55 552313 5-9-3 2′-MOE 66 552367 6-9-2 2′-MOE 67 552314 5-9-3 2′-MOE 56 552368 6-9-2 2′-MOE 41 552315 5-9-3 2′-MOE 46 552369 6-9-2 2′-MOE 52 552316 5-9-3 2′-MOE 55 552370 6-9-2 2′-MOE 35 552317 5-9-3 2′-MOE 53 552371 6-9-2 2′-MOE 58 552318 5-9-3 2′-MOE 59 552372 6-9-2 2′-MOE 68 552319 5-9-3 2′-MOE 56 552373 6-9-2 2′-MOE 63 552320 5-9-3 2′-MOE 62 552374 6-9-2 2′-MOE 70 552321 5-9-3 2′-MOE 63 552375 6-9-2 2′-MOE 64 552322 5-9-3 2′-MOE 52 552376 6-9-2 2′-MOE 58 552323 5-9-3 2′-MOE 45 552377 6-9-2 2′-MOE 42 552324 5-9-3 2′-MOE 49 552378 6-9-2 2′-MOE 37 552325 5-9-3 2′-MOE 48 552379 6-9-2 2′-MOE 57 552326 5-9-3 2′-MOE 50 552380 6-9-2 2′-MOE 48 552327 5-9-3 2′-MOE 13 552381 6-9-2 2′-MOE 22 552328 5-9-3 2′-MOE 9 552382 6-9-2 2′-MOE 20 552329 5-9-3 2′-MOE 18 552383 6-9-2 2′-MOE 18

Example 53 Dose-Dependent Antisense Inhibition of Target-Z mRNA in HepG2 Cells

Antisense oligonucleotides from the study described in Example 52 exhibiting in vitro inhibition of Target-Z mRNA were selected and tested at various doses in HepG2 cells. Cells were plated at a density of 28,000 cells per well and transfected using LipofectAMINE2000® with 9.26 nM, 27.78 nM, 83.33 nM, and 250.00 nM concentrations of antisense oligonucleotide, as specified in Table 97. After a treatment period of approximately 16′ hours, RNA was isolated from the cells and Target-Z mRNA levels were measured by quantitative real-time PCR. Target-Z primer probe set RTS3371 was used to measure mRNA levels. Target-Z mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-Z, relative to untreated control cells.

As illustrated in Table 97, Target-Z mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. ‘n/a’ indicates that the data for that dosage is not available.

TABLE 97 Dose-dependent antisense inhibition of human Target-Z in HepG2 cells 9.2593 27.7778 83.3333 250.0 ISIS No nM nM nM nM 146786 10 43 74 89 509934 12 31 52 79 509959 4 24 49 67 510100 11 28 60 77 510124 3 11 13 41 551926 1 26 51 76 551958 15 17 56 82 551987 4 40 65 81 551990 7 55 78 91 551993 15 30 70 80 551994 0 30 39 58 551995 6 41 73 85 551996 13 47 71 85 551997 16 38 68 89 551998 4 36 69 85 551999 10 31 67 86 552000 0 17 61 78 552006 6 37 74 89 552009 1 5 39 60 552013 0 28 3 72 552014 0 26 32 77 552018 6 27 63 81 552019 15 34 65 90 552020 2 35 65 91 552021 4 11 53 82 552022 6 35 57 79 552023 11 33 59 81 552024 15 43 69 91 552025 17 35 69 87 552026 14 26 66 86 552027 3 46 62 88 552028 9 43 58 78 552029 8 40 72 89 552030 18 48 77 92 552031 0 38 66 89 552032 42 48 80 88 552033 2 40 64 84 552034 6 40 70 81 552039 2 33 56 83 552044 19 30 63 84 552046 4 21 47 77 552050 15 44 70 92 552051 8 33 69 90 552052 17 38 71 91 552053 0 40 59 86 552054 7 15 58 75 552056 19 62 86 92 552057 11 33 69 86 552058 30 55 79 90 552059 11 25 69 90 552060 9 32 61 86 552061 6 40 69 88 552062 22 48 75 89 552064 23 49 69 90 552065 10 8 69 86 552069 11 4 28 60 552073 9 31 62 78 552075 21 18 33 65 552077 0 17 40 72 552079 1 12 44 70 552080 3 12 34 69 552082 13 29 66 87 552083 24 54 69 88 552084 10 25 48 82 552085 28 35 64 85 552086 0 24 65 84 552088 33 53 77 93 552089 0 41 69 92 552090 17 35 70 87 552091 13 31 69 89 552092 6 23 66 89 552093 0 17 61 89 552094 12 38 65 88 552095 20 42 73 88 552096 n/a 39 66 91 552097 24 43 67 88 552098 0 24 56 85 552101 3 13 28 61 552147 11 27 58 80 552160 20 25 69 89 552163 0 21 22 53 552176 16 11 40 66 552192 7 38 78 89 552222 0 24 65 79 552247 0 38 69 86 552255 5 27 69 81 552301 5 38 65 86 552309 8 26 62 85 552312 0 4 32 62 552347 2 15 38 75 552348 12 40 42 65 552354 10 35 44 76 552361 2 25 55 74 552363 20 36 54 76 552374 7 4 38 76 552379 0 12 24 46 552403 8 27 54 76 552408 2 25 44 77 552409 6 31 56 80 552418 0 30 72 84 552420 9 34 53 81 552442 4 23 46 56 552466 0 23 56 79 552474 11 34 66 87 552477 11 22 44 64 552530 25 37 73 87 552559 9 13 29 51

Example 54 Efficacy of Antisense Oligonucleotides Targeting Target-Z in Transgenic Mice

Target-Z transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for their efficacy in this model.

Treatment

Groups of 12 mice each were injected subcutaneously twice a week for 4 weeks with 50 mg/kg of ISIS 510106, ISIS 510116, ISIS 505347, or ISIS 509934. A control group of 12 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and livers were harvested for further analysis.

DNA and RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of Target-Z DNA, using primer probe sets RTS3370, RTS3371, and RTS3372. The DNA levels were normalized to picogreen. Target-Z RNA samples were also assayed with primer probe sets RTS3370 and RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. The data is presented in Table 98, expressed as percent inhibition compared to the control group. As shown in Table 98, most of the antisense oligonucleotides achieved reduction of Target-Z DNA and RNA over the PBS control. Results are presented as percent inhibition of Target-Z mRNA or DNA, relative to control.

TABLE 98 Percent inhibition of Target-Z RNA and DNA in the liver of transgenic mice % % % % % % inhibition inhibition inhibition inhibition inhibition inhibition DNA DNA DNA RNA RNA RNA ISIS No (RTS3370) (RTS3371) (RTS3372) (RTS3370) (RTS3371) (RTS3372) 510106 0 0 51 0 0 12 510116 68 79 68 49 54 66 505347 72 79 75 54 28 30 509934 93 95 94 72 75 92

Example 55 Efficacy of Antisense Oligonucleotides Targeting Target-Z in Transgenic Mice

Target-Z transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for their efficacy in this model.

Treatment

Groups of 6 mice each were injected subcutaneously twice a week for 4 weeks with 50 mg/kg of ISIS 146779, ISIS 505358, ISIS 146786, ISIS 509974, ISIS 509958, or ISIS 509959. A control group of 10 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and livers were harvested for further analysis.

DNA and RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of Target-Z DNA, using primer probe sets RTS3370. The DNA levels were normalized to picogreen. Target-Z RNA samples were also assayed with primer probe sets RTS3370 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. The data is presented in Table 99, expressed as percent inhibition compared to the control group. As shown in Table 99, most of the antisense oligonucleotides achieved reduction of Target-Z DNA and RNA over the PBS control. Results are presented as percent inhibition of Target-Z mRNA or DNA, relative to control.

TABLE 99 Percent inhibition of Target-Z RNA and DNA in the liver of transgenic mice % % inhibition inhibition ISIS No DNA RNA 146779 39  5 505358 84 77 146786 83 73 509974 56 28 509958 82 29 509959 54 30

Example 56 Efficacy of Antisense Oligonucleotides Targeting Target-Z in Transgenic Mice

Transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for their efficacy in this model.

Treatment

Groups of 6 mice each were injected subcutaneously twice a week for 4 weeks with 25 mg/kg of ISIS 146786, ISIS 552176, and ISIS 552073. One group of 10 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

DNA and RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of Target-Z DNA, using primer probe set RTS3371. The DNA levels were normalized to picogreen. Target-Z RNA samples were also assayed with primer probe set RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. The data is presented in Table 100. Serum DNA samples were analyzed after the study period. The data is presented in Table 101, expressed relative to the levels measured in the control group. As shown in Tables 100 and 101, the antisense oligonucleotides achieved reduction of Target-Z DNA and RNA over the PBS control. Results are presented as percent inhibition of Target-Z mRNA or DNA, relative to control.

TABLE 100 Percent inhibition of Target-Z RNA and DNA in transgenic mice Dose % inhibition % inhibition ISIS No (mg/kg/wk) of RNA of DNA 146786 50 81 91 552073 50 39 22 552176 50 55 56

TABLE 101 Serum levels of Target-Z DNA in transgenic mice, relative to control levels Dose Post-dose ISIS No (mg/kg/wk) DNA levels 146786 50 0.1 552073 50 2.9 552176 50 2.1

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma concentrations of ALT were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.) (Nyblom, H. et al., Alcohol & Alcoholism 39: 336-339, 2004; Tietz N W (Ed): Clinical Guide to Laboratory Tests, 3rd ed. W. B. Saunders, Philadelphia, PA, 1995). The results are presented in Table 102 expressed in IU/L. Both the ISIS oligonucleotides were considered tolerable in the mice, as demonstrated by their liver transaminase profile.

TABLE 102 ALT levels (IU/L) of transgenic mice Dose (mg/kg/wk) ALT PBS — 77 ISIS 146786 50 21 ISIS 552073 50 19 ISIS 552176 50 27

Example 57 Efficacy of Antisense Oligonucleotides Targeting Target-Z in Transgenic Mice

Transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for their efficacy in this model.

Treatment

Groups of 6 mice each were injected subcutaneously twice a week for 4 weeks with 25 mg/kg of ISIS 146786, ISIS 552056, ISIS 552088, and ISIS 552309. One group of 10 mice was injected subcutaneously twice a week for 4 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

DNA and RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of Target-Z DNA, using primer probe set RTS3371. The DNA levels were normalized to picogreen. Target-Z RNA samples were also assayed with primer probe set RTS3371 after RT-PCR analysis. The mRNA levels were normalized to RIBOGREEN®. As shown in Table 103, the antisense oligonucleotides achieved reduction of Target-Z DNA and RNA over the PBS control. Results are presented as percent inhibition of Target-Z mRNA or DNA, relative to control.

TABLE 103 Percent inhibition of Target-Z DNA and RNA in transgenic mice % % Dose inhibition inhibition (mg/kg/wk) (RNA) (DNA) ISIS 146786 50 60 90 ISIS 552056 50 25 58 ISIS 552088 50 8 0 ISIS 552309 50 35 84

Example 58 Antisense Inhibition of Human Target-1 in HuVEC Cells

Antisense oligonucleotides were designed targeting a human Target-1 nucleic acid and were tested for their effect on human Target-1 mRNA expression in vitro. The chimeric antisense oligonucleotides presented in Tables 104 and 105 were either 2-10-2 cEt gapmers or 3-10-3 cEt gapmers. The internucleoside linkages throughout each gapmer was phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer were 5′-methylcytosines.

Cultured HuVEC cells at a density of 20,000 cells per well were transfected using electroporation with 1,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-1 mRNA levels were measured by quantitative real-time PCR. Target-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-1, relative to untreated control cells. All cEt gapmers and MOE gapmers were tested under the same conditions.

“Human Target start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Human Target stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in Table 104 is targeted to human Target-1 mRNA. Each gapmer listed in Table 105 is targeted to the human Target-1 genomic sequence, truncated from nucleotides 4185000 to 4264000. Throughout the Examples, oligonucleotides having the same Sequence Number have the same nucleobase sequence.

TABLE 104 Inhibition of human Target-1 mRNA levels by cEt and MOE antisense oligonucleotides targeted to Target-1 mRNA Human Human ISIS Start Stop Wing % NO Site Site Motif Chemistry inhibition 481353 240 255 3-10-3 cEt 78 481354 264 279 3-10-3 cEt 98 481579 265 278 2-10-2 cEt 91 481355 322 337 3-10-3 cEt 95 481580 323 336 2-10-2 cEt 76 481356 346 361 3-10-3 cEt 83 481357 375 390 3-10-3 cEt 97 481582 376 389 2-10-2 cEt 87 481358 403 418 3-10-3 cEt 85 481359 429 444 3-10-3 cEt 90 481361 474 489 3-10-3 cEt 90 481586 475 488 2-10-2 cEt 81 481363 511 526 3-10-3 cEt 84 481368 659 674 3-10-3 cEt 81 481371 709 724 3-10-3 cEt 83 481372 730 745 3-10-3 cEt 85 481597 731 744 2-10-2 cEt 80 481373 751 766 3-10-3 cEt 87 481374 788 803 3-10-3 cEt 92 481599 789 802 2-10-2 cEt 51 481376 868 883 3-10-3 cEt 82 481601 869 882 2-10-2 cEt 70 481377 884 899 3-10-3 cEt 85 481378 892 907 3-10-3 cEt 89 481379 955 970 3-10-3 cEt 91 481604 956 969 2-10-2 cEt 70 481380 963 978 3-10-3 cEt 73 481605 964 977 2-10-2 cEt 55 481382 1045 1060 3-10-3 cEt 81 481383 1053 1068 3-10-3 cEt 84 481384 1098 1113 3-10-3 cEt 76 481387 1225 1240 3-10-3 cEt 92 481612 1226 1239 2-10-2 cEt 86 481388 1269 1284 3-10-3 cEt 74 481390 1305 1320 3-10-3 cEt 92 481396 1480 1495 3-10-3 cEt 92 481621 1481 1494 2-10-2 cEt 74 481398 1542 1557 3-10-3 cEt 73 481399 1563 1578 3-10-3 cEt 73 481401 1589 1604 3-10-3 cEt 74 481405 1641 1656 3-10-3 cEt 75 481406 1691 1706 3-10-3 cEt 72 481409 1795 1810 3-10-3 cEt 86 481410 1825 1840 3-10-3 cEt 91 481412 1858 1873 3-10-3 cEt 90 481637 1859 1872 2-10-2 cEt 79 481413 1866 1881 3-10-3 cEt 80 481638 1867 1880 2-10-2 cEt 64 481414 1888 1903 3-10-3 cEt 69 481639 1889 1902 2-10-2 cEt 16 481415 1896 1911 3-10-3 cEt 88 481640 1897 1910 2-10-2 cEt 57 337332 1898 1917 5-10-5 MOE 63 481416 1901 1916 3-10-3 cEt 87 481641 1902 1915 2-10-2 cEt 68 337333 1903 1922 5-10-5 MOE 49 481417 1903 1918 3-10-3 cEt 97 481418 1904 1919 3-10-3 cEt 92 481642 1904 1917 2-10-2 cEt 67 481419 1905 1920 3-10-3 cEt 83 481643 1905 1918 2-10-2 cEt 58 481644 1906 1919 2-10-2 cEt 45 481420 1948 1963 3-10-3 cEt 94 481645 1949 1962 2-10-2 cEt 50 481421 2021 2036 3-10-3 cEt 86 481422 2036 2051 3-10-3 cEt 80 481425 2115 2130 3-10-3 cEt 78 481650 2116 2129 2-10-2 cEt 79 481426 2131 2146 3-10-3 cEt 80 481651 2132 2145 2-10-2 cEt 64 481427 2155 2170 3-10-3 cEt 75 481652 2156 2169 2-10-2 cEt 82 481428 2164 2179 3-10-3 cEt 77 481653 2165 2178 2-10-2 cEt 79 481429 2172 2187 3-10-3 cEt 84 481654 2173 2186 2-10-2 cEt 70 481430 2190 2205 3-10-3 cEt 67 481431 2206 2221 3-10-3 cEt 91 481433 2256 2271 3-10-3 cEt 73 481658 2257 2270 2-10-2 cEt 62 481434 2266 2281 3-10-3 cEt 73 345785 2267 2286 5-10-5 MOE 50 481659 2267 2280 2-10-2 cEt 51 481435 2269 2284 3-10-3 cEt 49 481660 2270 2283 2-10-2 cEt 54 481436 2275 2290 3-10-3 cEt 82 481661 2276 2289 2-10-2 cEt 76 481439 2371 2386 3-10-3 cEt 82 481664 2372 2385 2-10-2 cEt 89 481440 2387 2402 3-10-3 cEt 79 481445 2439 2454 3-10-3 cEt 70 481449 2503 2518 3-10-3 cEt 77 481452 2631 2646 3-10-3 cEt 71 481453 2681 2696 3-10-3 cEt 92 481678 2682 2695 2-10-2 cEt 78 481454 2702 2717 3-10-3 cEt 85 481679 2703 2716 2-10-2 cEt 69 481455 2722 2737 3-10-3 cEt 74 481457 2779 2794 3-10-3 cEt 88 481682 2780 2793 2-10-2 cEt 77 481459 2908 2923 3-10-3 cEt 76 481684 2909 2922 2-10-2 cEt 89 481460 2943 2958 3-10-3 cEt 83 481461 2969 2984 3-10-3 cEt 75 481686 2970 2983 2-10-2 cEt 70 481462 2984 2999 3-10-3 cEt 89 481687 2985 2998 2-10-2 cEt 80 481463 1001 3016 3-10-3 cEt R8 481688 3002 3015 2-10-2 cEt 13 481464 3016 3031 3-10-3 cEt 97 481689 3017 3030 2-10-2 cEt 40 481466 3047 3062 3-10-3 cEt 74 481691 3048 3061 2-10-2 cEt 77 481467 3097 3112 3-10-3 cEt 74 481692 3098 3111 2-10-2 cEt 74 481468 3112 3127 3-10-3 cEt 71 481695 3462 3475 2-10-2 cEt 83 481472 3491 3506 3-10-3 cEt 76 481697 3492 3505 2-10-2 cEt 63 481474 3521 3536 3-10-3 cEt 80 481475 3536 3551 3-10-3 cEt 93 481700 3537 3550 2-10-2 cEt 89 481476 3551 3566 3-10-3 cEt 92 481701 3552 3565 2-10-2 cEt 60 481477 3567 3582 3-10-3 cEt 95 481702 3568 3581 2-10-2 cEt 89 481478 3585 3600 3-10-3 cEt 84 481479 3600 3615 3-10-3 cEt 80 481485 3717 3732 3-10-3 cEt 90 481710 3718 3731 2-10-2 cEt 88 481486 3730 3745 3-10-3 cEt 75 481711 3731 3744 2-10-2 cEt 74 481490 3833 3848 3-10-3 cEt 78 481715 3834 3847 2-10-2 cEt 79 481491 3848 3863 3-10-3 cEt 70 481716 3849 3862 2-10-2 cEt 68 481495 3940 3955 3-10-3 cEt 92 481498 3992 4007 3-10-3 cEt 90 481723 3993 4006 2-10-2 cEt 49 481499 4007 4022 3-10-3 cEt 43 481724 4008 4021 2-10-2 cEt 17 481500 4022 4037 3-10-3 cEt 92 481501 4048 4063 3-10-3 cEt 91 481502 4063 4078 3-10-3 cEt 85 481727 4064 4077 2-10-2 cEt 70 481510 4237 4252 3-10-3 cEt 95 481735 4238 4251 2-10-2 cEt 22 481513 4290 4305 3-10-3 cEt 85 481738 4291 4304 2-10-2 cEt 70 481514 4305 4320 3-10-3 cEt 85 481719 4306 4319 2-10-2 cEt 60 481515 4325 4340 3-10-3 cEt 88 481740 4326 4339 2-10-2 cEt 71 481516 4364 4379 3-10-3 cEt 78 481741 4365 4378 2-10-2 cEt 80 481517 4394 4409 3-10-3 cEt 87 481742 4395 4408 2-10-2 cEt 64 481518 4425 4440 3-10-3 cEt 67 481743 4426 4439 2-10-2 cEt 75 481519 4437 4452 3-10-3 cEt 29 481744 4438 4451 2-10-2 cEt 69 481520 4439 4454 3-10-3 cEt 73 481745 4440 4453 2-10-2 cEt 74 481521 4459 4474 3-10-3 cEt 86 481746 4460 4473 2-10-2 cEt 67 481522 4474 4489 3-10-3 cEt 92 481747 4475 4488 2-10-2 cEt 95 481523 4489 4504 3-10-3 cEt 95 481524 4530 4545 3-10-3 cEt 70 481749 4531 4544 2-10-2 cEt 70 481525 4541 4556 3-10-3 cEt 93 481750 4542 4555 2-10-2 cEt 94 481526 4543 4558 3-10-3 cEt 82 481528 4579 4594 3-10-3 cEt 77 481753 4580 4593 2-10-2 cEt 71 481530 4630 4645 3-10-3 cEt 87 481755 4631 4644 2-10-2 cEt 84 481532 4664 4679 3-10-3 cEt 65 481757 4665 4678 2-10-2 cEt 81 481533 4666 4681 3-10-3 cEt 80 481758 4667 4680 2-10-2 cEt 62 481534 4693 4708 3-10-3 cEt 79 481759 4694 4707 2-10-2 cEt 74 481535 4767 4782 3-10-3 cEt 78 481760 4768 4781 2-10-2 cEt 78 481536 4782 4797 3-10-3 cEt 91 481761 4783 4796 2-10-2 cEt 78 481537 4830 4845 3-10-3 cEt 84 481538 4844 4859 3-10-3 cEt 92 481763 4845 4858 2-10-2 cEt 96 481541 4934 4949 3-10-3 cEt 71

TABLE 105 Inhibition of human Target-1 mRNA levels by cEt and MOE antisense oligonucleotides targeted to Target-1 Genomic Sequence Human Human ISIS Start Stop Wing % NO Site Site Motif Chemistry inhibition 481543  1996  2011 3-10-3 cEt 84 481768  1997  2010 2-10-2 cEt 95 481546  2113  2128 3-10-3 cEt 70 481771  2114  2127 2-10-2 cEt 75 481547  2121  2136 3-10-3 cEt 87 481548  2705  2720 3-10-3 cEt 78 481549  6476  6491 3-10-3 cEt 96 481774  6477  6490 2-10-2 cEt 56 481553 10364 10379 3-10-3 cEt 96 481554 15469 15484 3-10-3 cEt 86 481779 15470 15483 2-10-2 cEt 60 481555 24588 24603 3-10-3 cEt 73 481780 24589 24602 2-10-2 cEt 60 481353 40968 40983 3-10-3 cEt 78 481354 40992 41007 3-10-3 cEt 98 481579 40993 41006 2-10-2 cEt 91 481355 41050 41065 3-10-3 cEt 95 481580 41051 41064 2-10-2 cEt 76 481356 41074 41089 3-10-3 cEt 83 481581 41075 41088 2-10-2 cEt 31 481357 42778 42793 3-10-3 cEt 97 481582 42779 42792 2-10-2 cEt 87 481358 42806 42821 3-10-3 cEt 85 481359 42832 42847 3-10-3 cEt 90 481360 42862 42877 3-10-3 cEt 75 481585 42863 42876 2-10-2 cEt 77 481361 42877 42892 3-10-3 cEt 90 481586 42878 42891 2-10-2 cEt 81 481368 50122 50137 3-10-3 cEt 81 481559 50668 50683 3-10-3 cEt 72 481784 50669 50682 2-10-2 cEt 79 481371 50673 50688 3-10-3 cEt 83 481372 50694 50709 3-10-3 cEt 85 481597 50695 50708 2-10-2 cEt 80 481373 50715 50730 3-10-3 cEt 87 481376 51705 51720 3-10-3 cEt 82 481601 51706 51719 2-10-2 cEt 70 481378 51905 51970 1-10-1 cEt 89 481603 51906 51919 2-10-2 cEt 60 481379 51968 51983 3-10-3 cEt 91 481604 51969 51982 2-10-2 cEt 70 481380 51976 51991 3-10-3 cEt 73 481382 55443 55458 3-10-3 cEt 81 481383 55451 55466 3-10-3 cEt 84 481384 55496 55511 3-10-3 cEt 76 481387 55748 55763 3-10-3 cEt 92 481612 55749 55762 2-10-2 cEt 86 481388 55792 55807 3-10-3 cEt 74 481390 57969 57984 3-10-3 cEt 92 481396 60034 60049 3-10-3 cEt 92 481621 60035 60048 2-10-2 cEt 74 481398 63306 63321 3-10-3 cEt 73 481399 63327 63342 3-10-3 cEt 73 481401 63353 63368 3-10-3 cEt 74 481405 64459 64474 3-10-3 cEt 75 481409 64729 64744 3-10-3 cEt 86 481410 64759 64774 3-10-3 cEt 91 481411 65859 65874 3-10-3 cEt 72 481412 65877 65892 3-10-3 cEt 90 481637 65878 65891 2-10-2 cEt 79 481413 65885 65900 3-10-3 cEt 80 481638 65886 65899 2-10-2 cEt 64 481566 66127 66142 3-10-3 cEt 62 481791 66128 66141 2-10-2 cEt 73 481415 66133 66148 3-10-3 cEt 88 481640 66134 66147 2-10-2 cEt 57 337332 66135 66154 5-10-5 MOE 63 481416 66138 66153 3-10-3 cEt 87 481641 66139 66152 2-10-2 cEt 68 337333 66140 66159 5-10-5 MOE 49 481417 66140 66155 3-10-3 cEt 97 481418 66141 66156 3-10-3 cEt 92 481642 66141 66154 2-10-2 cEt 67 481419 66142 66157 3-10-3 cEt 83 481420 66185 66200 3-10-3 cEt 94 481645 66186 66199 2-10-2 cEt 50 481421 66374 66389 3-10-3 cEt 86 481422 66389 66404 3-10-3 cEt 80 481423 66430 66445 3-10-3 cEt 69 481424 66446 66461 3-10-3 cEt 70 481425 66468 66483 3-10-1 cEt 78 481650 66469 66482 2-10-2 cEt 79 481426 66993 67008 3-10-3 cEt 80 481651 66994 67007 2-10-2 cEt 64 481427 67017 67032 3-10-3 cEt 75 481652 67018 67031 2-10-2 cEt 82 481428 67026 67041 3-10-3 cEt 77 481653 67027 67040 2-10-2 cEt 79 481429 67034 67049 3-10-3 cEt 84 481654 67035 67048 2-10-2 cEt 70 481430 67052 67067 3-10-3 cEt 67 481431 67068 67083 3-10-3 cEt 91 481433 67118 67133 3-10-3 cEt 73 481658 67119 67132 2-10-2 cEt 62 481434 67128 67143 3-10-3 cEt 73 345785 67129 67148 5-10-5 MOE 50 481659 67129 67142 2-10-2 cEt 51 481435 67131 67146 3-10-3 cEt 49 481660 67132 67145 2-10-2 cEt 54 481436 67137 67152 3-10-3 cEt 82 481661 67138 67151 2-10-2 cEt 76 481568 72290 72305 3-10-3 cEt 85 481793 72291 72304 2-10-2 cEt 93 481569 72430 72445 3-10-3 cEt 62 481794 72431 72444 2-10-2 cEt 81 481570 72438 72453 3-10-3 cEt 79 481440 72586 72601 3-10-3 cEt 79 481443 72622 72637 3-10-3 cEt 78 481444 72630 72645 3-10-3 cEt 66 481445 72638 72653 3-10-3 cEt 70 481670 72639 72652 2-10-2 cEt 60 481449 73690 73705 3-10-3 cEt 77 481452 73818 73833 3-10-3 cEt 71 481453 73868 73883 3-10-3 cEt 92 481678 73869 73882 2-10-2 cEt 78 481454 73889 73904 3-10-3 cEt 85 481679 73890 73903 2-10-2 cEt 69 481455 73909 73924 3-10-3 cEt 74 481457 73966 73981 3-10-3 cEt 88 481682 73967 73980 2-10-2 cEt 77 481459 74095 74110 3-10-3 cEt 76 481684 74096 74109 2-10-2 cEt 89 481460 74130 74145 3-10-3 cEt 83 481685 74131 74144 2-10-2 cEt 16 481461 74156 74171 3-10-3 cEt 75 481686 74157 74170 2-10-2 cEt 70 481462 74171 74186 3-10-3 cEt 89 481687 74172 74185 2-10-2 cEt 80 481463 74188 74203 3-10-3 cEt 88 481688 74189 74202 2-10-2 cEt 13 481464 74203 74218 3-10-3 cEt 97 481689 74204 74217 2-10-2 cEt 40 481466 74234 74249 3-10-3 cEt 74 481691 74235 74248 2-10-2 cEt 77 481467 74284 74299 3-10-3 cEt 74 481692 74285 74298 2-10-2 cEt 74 481468 74299 74314 3-10-3 cEt 71 481695 74649 74662 2-10-2 cEt 83 481472 74678 74693 3-10-3 cEt 76 481697 74679 74692 2-10-2 cEt 63 481474 74708 74723 3-10-3 cEt 80 481475 74723 74738 3-10-3 cEt 93 481700 74724 74737 2-10-2 cEt 89 481476 74738 74753 3-10-3 cEt 92 481701 74739 74752 2-10-2 cEt 60 481477 74754 74769 3-10-3 cEt 95 481702 74755 74768 2-10-2 cEt 89 481478 74772 74787 3-10-3 cEt 84 481479 74787 74802 3-10-3 cEt 80 481485 74904 74919 3-10-3 cEt 90 481710 74905 74918 2-10-2 cEt 88 481486 74917 74932 3-10-3 cEt 75 481711 74918 74931 2-10-2 cEt 74 481487 74933 74948 3-10-3 cEt 66 481490 75020 75035 3-10-3 cEt 78 481715 75021 75034 2-10-2 cEt 79 481491 75035 75050 3-10-3 cEt 70 481716 75036 75049 2-10-2 cEt 68 481492 75050 75065 3-10-3 cEt 61 481495 75127 75142 3-10-3 cEt 92 481720 75128 75141 2-10-2 cEt 63 481498 75179 75194 3-10-3 cEt 90 481500 75209 75224 3-10-3 cEt 92 481725 75210 75223 2-10-2 cEt 88 481501 75235 75250 3-10-3 cEt 91 481502 75250 75265 3-10-3 cEt 85 481727 75251 75264 2-10-2 cEt 70 481510 75424 75439 3-10-3 cEt 95 481735 75425 75438 2-10-2 cEt 22 481513 75477 75492 3-10-3 cEt 85 481738 75478 75491 2-10-2 cEt 70 481514 75492 75507 3-10-3 cEt 85 481739 75493 75506 2-10-2 cEt 60 481515 75512 75527 3-10-3 cEt 88 481740 75513 75526 2-10-2 cEt 71 481516 75551 75566 3-10-3 cEt 78 481741 75552 75565 2-10-2 cEt 80 481517 75581 75596 3-10-3 cEt 87 481742 75582 75595 2-10-2 cEt 64 481518 75612 75627 3-10-3 cEt 67 481743 75613 75626 2-10-2 cEt 75 481744 75625 75638 2-10-2 cEt 69 481520 75626 75641 3-10-3 cEt 73 481745 75627 75640 2-10-2 cEt 74 481521 75646 75661 3-10-3 cEt 86 481746 75647 75660 2-10-2 cEt 67 481522 75661 75676 3-10-3 cEt 92 481747 75662 75675 2-10-2 cEt 95 481523 75676 75691 3-10-3 cEt 95 481524 75717 75732 3-10-3 cEt 70 481749 75718 75731 2-10-2 cEt 70 481525 75728 75743 3-10-3 cEt 93 481750 75729 75742 2-10-2 cEt 94 481526 75730 75745 3-10-3 cEt 82 481528 75766 75781 3-10-3 cEt 77 481753 75767 75780 2-10-2 cEt 71 481530 75817 75832 3-10-3 cEt 87 481755 75818 75831 2-10-2 cEt 84 481757 75852 75865 2-10-2 cEt 81 481533 75853 75868 3-10-3 cEt 80 481758 75854 75867 2-10-2 cEt 62 481534 75880 75895 3-10-3 cEt 79 481759 75881 75894 2-10-2 cEt 74 481535 75954 75969 3-10-3 cEt 78 481760 75955 75968 2-10-2 cEt 78 481536 75969 75984 3-10-3 cEt 91 481761 75970 75983 2-10-2 cEt 78 481537 76017 76032 3-10-3 cEt 84 481538 76031 76046 3-10-3 cEt 92 481763 76032 76045 7-10-7 cEt 96 481539 76047 76062 3-10-3 cEt 19 481541 76121 76136 3-10-3 cEt 71

Example 59 Antisense Inhibition of Murine Target-1 in b.END Cells

Antisense oligonucleotides tested in the study described in Example 58 were also tested for their effects on Target-1 mRNA in b.END cells. Cultured b.END cells at a density of 20,000 cells per well were transfected using electroporation with 7,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-1 mRNA levels were measured by quantitative real-time PCR. Target-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®.

Certain sequences complementary to the Target-I mouse gene sequence showed good inhibition in b.END cells. Results are presented in Table 106 as percent inhibition of Target-1, relative to untreated control cells. The human oligonucleotides in Table 106 were compared to the mouse Target-lgenomic sequence. “Mouse Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the murine sequence. “Mouse Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted murine sequence.

TABLE 106 Inhibition of human Target-1 mRNA levels by certain antisense oligonucleotides complementary to Murine Target-1 ISIS Mouse Start Stop Mouse % NO Site Site inhibition 481549 5283 5298 96 481553 9913 9928 94 481768 3189 3202 91 481356 30356 30371 83 481548 4045 4060 82 481554 14662 14677 82 481426 48328 48343 82 481580 30333 30346 81 481412 47413 47428 81 481417 47636 47651 81 481418 47637 47652 80 481355 30332 30347 79 481396 43120 43135 79 481416 47634 47649 79 481420 47681 47696 79 481358 32842 32857 78 481363 33520 33535 78 481570 51870 51885 78 481382 37857 37872 77 481378 36560 36575 76 481431 48403 48418 76 481453 53034 53049 76 481621 43121 43134 75 481641 47635 47648 75 481637 47414 47427 74 481380 36631 36646 73 481574 53000 53015 73 481601 36392 36405 71 481419 47638 47653 71 481371 35938 35953 70 481642 47637 47650 70 481542 3180 3195 69 481547 3313 3328 69 481772 3314 3327 69 481362 32929 32944 69 481653 48362 48375 69 481786 38812 38825 68 481415 47629 47644 68 481543 3188 3203 67 481793 51714 51727 67 481443 52060 52075 67 481684 53229 53242 67 481398 45226 45241 66 481560 36394 36409 65 481643 47638 47651 65 481430 48387 48402 65 481440 52024 52039 65

Example 60 Tolerability of Antisense Oligonucleotides Targeting Target-1 in BALB/c Mice

Forty antisense oligonucleotides exhibiting a high level of activity, selected from among the 452 compounds evaluated in Example 58, were further tested for in vivo tolerability.

Groups of 2-4 male BALB/c mice were injected subcutaneously twice a week for 3 weeks with 25 mg/kg of ISIS antisense oligonucleotides. One group of 4 male BALB/c mice was injected subcutaneously twice a week tbr 3 weeks with PBS. This group of mice was utilized as a control group to which the treatment groups were compared. One day after the last dose, body weights were taken, mice were euthanized, and organs and plasma were harvested for further analysis.

The body weights of the mice were measured pre-dose and at the end of the treatment period. Percent increase over the initial body weight was calculated. Liver, spleen, and kidney weights were measured at the end of the study and were compared to PBS treated mice.

To evaluate the effect of ISIS oligonucleotides on metabolic function, plasma concentrations of transaminases and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma concentrations of ALT (alanine transaminase), AST (aspartate transaminase), and BUN were measured.

Among the forty antisense oligonucleotides tested, certain antisense oligonucleotides, including ISIS 481374, ISIS 481390, ISIS 481420, ISIS 481431, ISIS 481453, ISIS 481464, ISIS 481475, ISIS 481495, ISIS 481500, ISIS 481501, ISIS 481525, ISIS 481548, ISIS 481549, ISIS 481597, ISIS 481695, ISIS 481700, ISIS 481702, ISIS 481710, ISIS 481725, ISIS 481750, and ISIS 481763 met tolerability thresholds for body weight, organ weight, ALT, AST, and BUN parameters.

Example 61 Dose-Dependent Antisense Inhibition of Human Target-1 in HuVEC Cells

Gapmers from Examples 58 and 59 exhibiting in vitro inhibition of Target-1 were tested at various doses in HuVEC cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 31.25 nM, 62.5 nM, 125 nM, 250 nM, 500 nM, and 1,0000 nM concentrations of antisense oligonucleotide, as specified in Table 107. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-1 mRNA levels were measured by quantitative real-time PCR. Target-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-1, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented in Table 107 and was calculated by plotting the concentrations of oligonucleotides used versus the percent inhibition of Target-1 mRNA expression achieved at each concentration and noting the concentration of oligonucleotide at which 50% inhibition of Target-1 mRNA expression was achieved compared to the control.

TABLE 107 Dose-dependent antisense inhibition of human Target-1 in HuVEC cells using electroporation 31.25 62.5 125.0 250.0 500.0 1000.0 IC₅₀ ISIS No nM nM nM nM nM nM (μM) 481355 19 15 36 61 75 89 0.18 481374 25 42 52 72 82 88 0.10 481390 17 37 44 60 73 86 0.15 481420 23 20 40 60 81 92 0.16 481453 21 37 52 69 79 88 0.12 481464 57 73 81 90 94 94 <0.03 481475 22 46 54 78 83 92 0.10 481500 25 37 42 75 83 90 0.12 481501 32 57 69 82 94 94 0.05 481523 35 60 74 85 90 93 0.04 481525 36 53 60 79 89 92 0.06 481549 0 16 60 81 90 96 0.15 481554 0 15 28 49 70 86 0.25 481597 8 18 39 48 64 83 0.24 481695 15 27 39 50 64 80 0.22 481700 0 17 44 58 80 88 0.20 481710 12 39 65 79 86 90 0.11 481715 11 26 32 44 53 69 0.36 481725 27 40 56 77 89 93 0.09 481750 7 24 46 63 83 89 0.16 481755 17 28 30 54 68 80 0.20 481768 7 21 27 44 67 85 0.26

Example 62 Dose-Dependent Antisense Inhibition of Target-1 Following Free Uptake of Antisense Oligonucleotide in SK-BR-3 Cells

Gapmers from Example 61 were tested at various doses in SK-BR-3 cells. Cells were plated at a density of 4,000 cells per well. Cells were incubated with 0.02 μM, 0.1 μM, 0.5 μM, 1 μM, 2.5 μM, and 10 μM concentrations of antisense oligonucleotide, as specified in Table 108. After approximately 24 hours, RNA was isolated from the cells and Target-1 mRNA levels were measured by quantitative real-time PCR. Target-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-1, relative to untreated control cells. The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented in Table 108.

TABLE 108 Dose-dependent antisense inhibition of human Target 1 by free-uptake of ISIS oligonucleotide by SK-BR-3 cells ISIS No 0.02 μM 0.1 μM 0.5 μM 1 μM 2.5 μM 10 μM IC₅₀ (μM) 481374 10 18 18 16 8 25 15.9 481390 0 10 11 12 40 72 3.2 481453 14 13 27 45 58 79 1.3 481464 23 32 57 70 85 93 0.5 481475 0 0 35 49 72 88 1.0 481500 7 9 26 45 49 75 1.7 481501 0 0 4 5 53 65 2.7 481523 9 24 56 67 83 92 0.5 481525 0 17 13 15 32 68 4.4 481549 0 0 0 16 33 54 8.2 481597 1 0 11 14 22 44 10.6 481695 0 0 0 0 0 0 — 481710 5 0 10 13 27 66 6.0 481725 29 45 47 39 39 63 2.6 481750 19 24 36 42 71 80 1.1 481763 30 38 51 63 81 89 0.6 481768 12 5 34 25 32 35 12.4

Example 63 Dose-Dependent Antisense Inhibition of Target-1 Following Free Uptake of Antisense Oligonucleotide in U251-MG Cells

Gapmers from Example 62 were further tested at various doses in U251-MG cells. Cells were plated at a density of 4,000 cells per well. Cells were incubated with 0.02 μM, 0.1 μM, 0.5 μM, 1 μM, 2.5 μM, and 10 μM concentrations of antisense oligonucleotide, as specified in Table 109. After approximately 24 hours, RNA was isolated from the cells and Target-1 mRNA levels were measured by quantitative real-time PCR. Target-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-1, relative to untreated control cells. The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented in Table 109.

TABLE 109 Dose-dependent antisense inhibition of Target-1 mRNA levels by free-uptake of ISIS oligonucleotide by U251-MG cells ISIS No 0.02 μM 0.1 μM 0.5 μM 1 μM 2.5 μM 10 μM IC₅₀ (μM) 481374 0 0 10 0 12 25 15.7 481390 0 4 10 8 16 31 13.9 481453 4 3 15 16 20 42 11.0 481464 13 11 41 42 54 79 1.3 481475 3 13 26 37 41 67 2.6 481500 2 12 14 12 25 38 11.7 481501 0 0 2 1 14 47 10.3 481523 22 27 39 45 63 83 1.1 481525 1 1 17 17 35 60 6.3 481549 0 0 0 0 9 29 14.5 481597 3 3 12 18 18 47 10.1 481695 0 14 12 22 25 33 12.9 481710 0 0 0 0 6 23 16.8 481725 0 0 5 7 20 38 11.8 481750 4 15 18 18 17 33 13.2 481763 15 16 25 36 36 64 3.2 481768 22 16 18 22 21 37 12.2

Example 64 Dose-Dependent Antisense Inhibition of Target-1 Following Free Uptake of Antisense Oligonucleotide in U251-MG Cells

ISIS 481464 and ISIS 481549, from the studies described above, were further tested at different doses in U251-MG cells. Cells were plated at a density of 4,000 cells per well. Cells were incubated with 0.1 μM, 1 μM, 5 μM, 10 μM, and 20 μM concentrations of antisense oligonucleotide, as specified in Table 110. After approximately 24 hours, RNA was isolated from the cells and Target-1 mRNA levels were measured by quantitative real-time PCR. Target-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-1, relative to untreated control cells.

TABLE 110 Dose-dependent antisense inhibition of Target-1 mRNA levels by free-uptake of ISIS oligonucleotide by U251-MG cells ISIS No 0.1 μM 1 μM 5 μM 10 μM 20 μM IC₅₀ (μM) 481464 0 30 69 80 79 2.3 481549 0 0 26 35 38 >20

Example 65 Dose-Dependent Antisense Inhibition of Target-1 Following Free Uptake of Antisense Oligonucleotide in MDA-MB-231 Cells

ISIS 481464 and ISIS 481549 were further tested at different doses in MDA-MB-231 cells. Cells were plated at a density of 4,000 cells per well. Cells were incubated with 0.02 μM, 0.2 μM, 1.0 μM, 5.0 μM, and 10.0 μM concentrations of antisense oligonucleotide, as specified in Table 111. After approximately 24 hours, RNA was isolated from the cells and Target-1 mRNA levels were measured by quantitative real-time PCR. Target-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-1, relative to untreated control cells.

TABLE 111 Dose-dependent antisense inhibition of Target-1 mRNA levels by free-uptake of ISIS oligonucleotide by MDA-MB-231 cells ISIS No 0.02 μM 0.2 μM 1.0 μM 5.0 μM 10.0 μM IC₅₀ (μM) 481464 0 25 71 85 87 0.6 481549 0 2 33 49 66 4.4

Example 66 Dose-Dependent Antisense Inhibition of Target-1 Following Free Uptake of Antisense Oligonucleotide in A431 Cells

ISIS 481464 and ISIS 481549 were further tested at different doses in A431 cells. Cells were plated at a density of 4,000 cells per well. Cells were incubated with 0.02 μM, 0.2 μM, 1.0 μM, 5.0 μM, and 10.0 μM concentrations of antisense oligonucleotide, as specified in Table 112. After approximately 24 hours, RNA was isolated from the cells and Target-1 mRNA levels were measured by quantitative real-time PCR. Target-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-1, relative to untreated control cells.

TABLE 112 Dose-dependent antisense inhibition of Target-1 mRNA levels by free-uptake of ISIS oligonucleotide by A431 cells ISIS No 0.02 μM 0.2 μM 1.0 μM 5.0 μM 10.0 μM IC₅₀ (μM) 481464 79 93 98 98 98 <0.02 481549 0 38 68 82 84 0.6

Example 67 Dose-Dependent Antisense Inhibition of Target-1 Following Free Uptake of Antisense Oligonucleotide in H460 Cells

ISIS 481464 and ISIS 481549 were further tested at different doses in H460 cells. Cells were plated at a density of 4,000 cells per well. Cells were incubated with 0.02 μM, 0.2 μM, 1.0 μM, 5.0 μM, and 10.0 μM concentrations of antisense oligonucleotide, as specified in Table 113. After approximately 24 hours, RNA was isolated from the cells and Target-1 mRNA levels were measured by quantitative real-time PCR. Target-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN Results are presented as percent inhibition of Target-1, relative to untreated control cells.

TABLE 113 Dose-dependent antisense inhibition of Target-1 mRNA levels by free-uptake of ISIS oligonucleotide by H460 cells ISIS No 0.02 μM 0.2 μM 1.0 μM 5.0 μM 10.0 μM IC₅₀ (μM) 481464 46 89 96 97 98 0.01 481549 8 53 78 96 98 0.23

Example 68 Antisense Inhibition of Human Target-1 in HuVEC Cells

Antisense oligonucleotides were designed targeting a human Target-1 nucleic acid and were tested for their effect on human Target-1 mRNA expression in vitro. Cultured HuVEC cells at a density of 20,000 cells per well were transfected using electroporation with 1,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-1 mRNA levels were measured by quantitative real-time PCR. Target-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-1, relative to untreated control cells.

The chimeric antisense oligonucleotides in Table 114 were designed as 3-10-3 MOE, deoxy, and cEt gapmers as indicated in the Table. The chemistry column of Table 114 presents the sugar motif of each gapmer, wherein ‘e’ indicates a 2′-MOE nucleoside, ‘k’ indicates a constrained ethyl (cEt) nucleoside, and ‘d’ indicates a 2′-deoxynucleoside. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5′-methylcytosines.

“Human Target start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Human Target stop site” indicates the 3′-most nucleoside to which the gapmer is targeted in the human gene sequence. Each gapmer listed in Table 114 is targeted to human Target-1 mRNA.

TABLE 114 Inhibition of human Target-1 mRNA levels by chimeric antisense oligonucleotides targeted to Target-1 mRNA Human Human Start Stop ISIS % Site Site No Chemistry inhibition 250 265 528204 e-e-e-d(10)-k-k-k 83 251 266 528205 e-e-e-d(10)-k-k-k 72 252 267 528206 e-e-e-d(10)-k-k-k 44 253 268 528207 e-e-e-d(10)-k-k-k 49 263 278 528208 e-e-e-d(10)-k-k-k 73 264 279 528209 e-e-e-d(10)-k-k-k 81 265 280 528210 e-e-e-d(10)-k-k-k 78 266 281 528211 e-e-e-d(10)-k-k-k 72 267 282 528212 e-e-e-d(10)-k-k-k 81 268 283 528213 e-e-e-d(10)-k-k-k 46 270 285 528214 e-e-e-d(10)-k-k-k 80 271 286 528215 e-e-e-d(10)-k-k-k 69 433 448 528269 e-e-e-d(10)-k-k-k 69 434 449 528270 e-e-e-d(10)-k-k-k 73 435 450 528271 e-e-e-d(10)-k-k-k 71 867 882 528378 e-e-e-d(10)-k-k-k 72 1146 1161 528501 e-e-e-d(10)-k-k-k 67 1147 1162 528502 e-e-e-d(10)-k-k-k 76 1153 1168 528503 e-e-e-d(10)-k-k-k 68 1154 1169 528504 e-e-e-d(10)-k-k-k 69 1155 1170 528505 e-e-e-d(10)-k-k-k 68 1206 1221 528518 e-e-e-d(10)-k-k-k 80 1207 1222 528519 e-e-e-d(10)-k-k-k 61 1208 1223 528520 e-e-e-d(10)-k-k-k 63 2699 2714 528833 e-e-e-d(10)-k-k-k 77 2980 2995 528845 e-e-e-d(10)-k-k-k 65 2981 2996 528846 e-e-e-d(10)-k-k-k 80 2982 2997 528847 e-e-e-d(10)-k-k-k 72 2983 2998 528848 e-e-e-d(10)-k-k-k 46 2984 2999 528849 e-e-e-d(10)-k-k-k 59 3001 3016 528850 e-e-e-d(10)-k-k-k 10 3008 3023 528851 e-e-e-d(10)-k-k-k 61 3010 3025 528852 e-e-e-d(10)-k-k-k 88 3012 3027 528853 e-e-e-d(10)-k-k-k 91 3016 3031 518349 e-e-e-d(10)-k-k-k 85 e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

Example 69 Dose-Dependent Antisense Inhibition of Human Target-1 in HuVEC Cells

Gapmers from the study described in Example 68, above, exhibiting in vitro inhibition of Target-1 were tested at various doses in HuVEC cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 23.4375 nM, 93.75 nM, 375.0 nM, and 1,500.0 nM concentrations of antisense oligonucleotide, as specified in Table 115. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-1 mRNA levels were measured by quantitative real-time PCR. Target-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-1, relative to untreated control cells.

TABLE 115 Dose-dependent antisense inhibition of human Target-1 in HuVEC cells 23.4375 93.75 375.0 1500.0 IC₅₀ ISIS No nM nM nM nM (μM) 518340 0 8 28 63 1.0 518349 13 30 68 90 0.2 528189 8 13 43 71 0.5 528204 4 24 53 79 0.3 528205 0 9 59 80 0.4 528208 0 19 56 84 0.3 528209 0 28 58 90 0.3 528210 0 16 49 87 0.3 528211 0 10 47 86 0.4 528212 0 16 42 83 0.4 528214 0 25 55 88 0.3 528215 3 16 53 82 0.3 528237 13 19 33 73 0.6 528245 3 16 53 78 0.4 528263 0 3 32 76 0.6 528264 9 0 19 50 >1.5 528268 0 7 25 63 1.0 528269 0 11 39 77 0.5 528270 5 9 48 79 0.4 528271 0 14 37 81 0.5 528327 0 0 26 72 0.8 528347 0 2 25 69 0.9 528357 0 17 36 69 0.6 528389 0 3 19 82 0.7 528501 0 17 40 69 0.6 528502 0 10 35 76 0.6 528503 3 1 38 70 0.7 528504 0 19 45 72 0.5 528505 0 7 41 73 0.6 528518 0 24 51 81 0.3 528534 0 8 32 72 0.7 528539 0 7 39 73 0.6 528557 0 9 26 53 >1.5 528565 4 12 31 57 1.3 528567 8 13 25 54 >1.5 528569 9 19 37 60 0.8 528574 5 17 32 62 0.9 528622 10 4 29 68 0.9 528623 0 13 24 62 1.1 528626 1 0 34 68 0.8 528627 22 19 30 64 1.0 528664 0 14 37 74 0.5 528675 0 10 28 62 1.0 528689 0 16 33 65 0.7 528691 0 3 34 61 0.9 528695 1 4 36 66 0.8 528697 3 15 39 72 0.5 528710 13 16 28 63 1.0 528711 8 13 14 62 >1.5 528726 0 8 36 72 0.6 528757 4 10 29 76 0.6 528758 1 5 28 62 1.1 528772 0 2 21 63 1.2 528773 9 8 28 70 0.8 528791 4 9 41 69 0.6 528822 0 0 40 46 >1.5 528833 0 23 47 82 0.4 528846 10 19 49 85 0.3 528847 0 19 45 75 0.4 528852 5 33 66 93 0.2 528853 19 46 77 95 0.1

Example 70 Antisense Inhibition of Human Target-1 in HuVEC Cells

Antisense oligonucleotides were designed targeting a human Target-1 nucleic acid and were tested for their effect on human Target-1 mRNA expression in vitro. The chimeric antisense oligonucleotides in Tables 116 and 117 are gapmers 16 or 17 nucleotides in length having various chemical modifications, as indicated in Tables 18 and 19, below. The chemistry column of Tables 116 and 117 provides the sugar motif of each gapmer, wherein ‘e’ indicates a 2′-MOE nucleoside, ‘k’ indicates a constrained ethyl (cEt) nucleoside, and ‘d’ indicates a 2′-deoxynucleoside. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5′-methylcytosines.

Cultured HuVEC cells at a density of 20,000 cells per well were transfected using electroporation with 1,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-1 mRNA levels were measured by quantitative real-time PCR. Target-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-1, relative to untreated control cells.

“Human Target start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Human Target stop site” indicates the 3′-most nucleoside to which the gapmer is targeted in the human gene sequence. Each gapmer listed in Table 116 is targeted to human Target-1 mRNA. Each gapmer listed in Table 117 is targeted to human Target-1 genomic sequence, truncated from nucleotides 4185000 to 4264000).

TABLE 116 Inhibition of human Target-1 mRNA levels by chimeric antisense oligonucleotides targeted to Target-1 mRNA 1 Human Human Start Stop ISIS % Site Site No Chemistry inhibition 728 743 530423 k-d(10)-k-e-k-e-e 70 729 745 530053 e-e-k-d(10)-k-e-k-e 84 729 744 530373 e-k-d(10)-k-e-k-e 85 730 745 530121 e-k-k-d(10)-k-k-e 77 730 745 530168 e-e-k-d(10)-k-k-e 75 730 745 530218 e-d-k-d(10)-k-k-e 61 730 745 530268 e-d-d-k-d(9)-k-k-e 76 730 745 530318 e-e-e-e-d(9)-k-k-e 27 786 801 530424 k-d(10)-k-e-k-e-e 42 787 803 530058 e-e-k-d(10)-k-e-k-e 73 787 802 530374 e-k-d(10)-k-e-k-e 71 788 803 530122 e-k-k-d(10)-k-k-e 80 788 803 530169 e-e-k-d(10)-k-k-e 72 788 803 530219 e-d-k-d(10)-k-k-e 55 788 803 530269 e-d-d-k-d(9)-k-k-e 76 788 803 530319 e-e-e-e-d(9)-k-k-e 30 897 912 528403 e-e-e-d(10)-k-k-k 72 962 977 528424 e-e-e-d(10)-k-k-k 42 1023 1038 528458 e-e-e-d(10)-k-k-k 70 1899 1914 530425 k-d(10)-k-e-k-e-e 73 1900 1916 530054 e-e-k-d(10)-k-e-k-e 75 1900 1915 530375 e-k-d(10)-k-e-k-e 77 1901 1916 530123 e-k-k-d(10)-k-k-e 86 1901 1916 530170 e-e-k-d(10)-k-k-e 87 1901 1916 530220 e-d-k-d(10)-k-k-e 74 1901 1916 530270 e-d-d-k-d(9)-k-k-e 87 1901 1916 530320 e-e-e-e-d(9)-k-k-e 17 1946 1961 530426 k-d(10)-k-e-k-e-e 55 1947 1963 530059 e-e-k-d(10)-k-e-k-e 73 1947 1962 530376 e-k-d(10)-k-e-k-e 77 1948 1963 530124 e-k-k-d(10)-k-k-e 79 1948 1963 530171 e-e-k-d(10)-k-k-e 69 1948 1963 530221 e-d-k-d(10)-k-k-e 64 1948 1963 530271 e-d-d-k-d(9)-k-k-e 73 1948 1963 530321 e-e-e-e-d(9)-k-k-e 44 2204 2219 530427 k-d(10)-k-e-k-e-e 43 2205 2221 530060 e-e-k-d(10)-k-e-k-e 77 2205 2220 530377 e-k-d(10)-k-e-k-e 66 2206 2221 530125 e-k-k-d(10)-k-k-e 65 2206 2221 530172 e-e-k-d(10)-k-k-e 59 2206 2221 530222 e-d-k-d(10)-k-k-e 48 2206 2221 530272 e-d-d-k-d(9)-k-k-e 63 2206 2221 530322 e-e-e-e-d(9)-k-k-e 55 2681 2696 530126 e-k-k-d(10)-k-k-e 70 2681 2696 530173 e-e-k-d(10)-k-k-e 62 2681 2696 530223 e-d-k-d(10)-k-k-e 44 2681 2696 530273 e-d-d-k-d(9)-k-k-e 63 2681 2696 530323 e-e-e-e-d(9)-k-k-e 63 3012 3027 530513 k-d(10)-k-e-k-e-e 88 3013 3028 530507 e-k-d(10)-k-e-k-e 86 3013 3028 530514 k-d(10)-k-e-k-e-e 80 3014 3029 530430 k-d(10)-k-e-k-e-e 87 3014 3029 530468 e-k-k-d(10)-k-k-e 81 3014 3029 530476 e-e-k-d(10)-k-k-e 82 3014 3029 530484 e-d-k-d(10)-k-k-e 74 3014 3029 530492 e-d-d-k-d(9)-k-k-e 83 3014 3029 530500 e-e-e-e-d(9)-k-k-e 56 3014 3029 530508 e-k-d(10)-k-e-k-e 83 3015 3031 530062 e-e-k-d(10)-k-e-k-e 94 3015 3030 530380 e-k-d(10)-k-e-k-e 94 3015 3030 530469 e-k-k-d(10)-k-k-e 91 3015 3030 530477 e-e-k-d(10)-k-k-e 87 3015 3030 530485 e-d-k-d(10)-k-k-e 87 3015 3030 530493 e-d-d-k-d(9)-k-k-e 81 3015 3030 530501 e-e-e-e-d(9)-k-k-e 74 3015 3030 530515 k-d(10)-k-e-k-e-e 87 3016 3031 481464 k-k-k-d(10)-k-k-k 93 3016 3031 518349 e-e-e-d(10)-k-k-k 58 3016 3031 519637 e-k-k-d(10)-k-k-e 96 3016 3031 530175 e-e-k-d(10)-k-k-e 93 3016 3031 530225 e-d-k-d(10)-k-k-e 85 3016 3031 530275 e-d-d-k-d(9)-k-k-e 91 3016 3031 530325 e-e-e-e-d(9)-k-k-e 91 3017 3032 530470 e-k-k-d(10)-k-k-e 91 3017 3032 530478 e-e-k-d(10)-k-k-e 87 3017 3032 530486 e-d-k-d(10)-k-k-e 84 3017 3032 530494 e-d-d-k-d(9)-k-k-e 60 3017 3032 530502 e-e-e-e-d(9)-k-k-e 64 3017 3032 530509 e-k-d(10)-k-e-k-e 80 3018 3033 530471 e-k-k-d(10)-k-k-e 83 3018 3033 530479 e-e-k-d(10)-k-k-e 74 3018 3033 530487 e-d-k-d(10)-k-k-e 71 3018 3033 530495 e-d-d-k-d(9)-k-k-e 68 3018 3033 530503 e-e-e-e-d(9)-k-k-e 53 3460 3476 530055 e-e-k-d(10)-k-e-k-e 45 3460 3475 530381 e-k-d(10)-k-e-k-e 74 3461 3476 530128 e-k-k-d(10)-k-k-e 52 3461 3476 530176 e-e-k-d(10)-k-k-e 66 3461 3476 530226 e-d-k-d(10)-k-k-e 51 3461 3476 530276 e-d-d-k-d(9)-k-k-e 70 3461 3476 530326 e-e-e-e-d(9)-k-k-e 52 3595 3610 530390 k-d(10)-k-e-k-e-e 83 3596 3611 530340 e-k-d(10)-k-e-k-e 89 3597 3612 528869 e-e-e-d(10)-k-k-k 83 3597 3612 530088 c-k-k-d(10)-k-k-e 90 3597 3612 530135 e-e-k-d(10)-k-k-e 91 3597 3612 530185 e-d-k-d(10)-k-k-e 85 3597 3612 530235 e-d-d-k-d(9)-k-k-e 28 3597 3612 530285 e-e-e-e-d(9)-k-k-e 86 3597 3612 530391 k-d(10)-k-e-k-e-e 79 3598 3614 530021 e-e-k-d(10)-k-e-k-e 87 3598 3613 530341 e-k-d(10)-k-e-k-e 88 3599 3614 530089 e-k-k-d(10)-k-k-e 71 3599 3614 530136 e-e-k-d(10)-k-k-e 66 3599 3614 530186 e-d-k-d(10)-k-k-e 51 3599 3614 530236 e-d-d-k-d(9)-k-k-e 74 3599 3614 530286 e-e-e-e-d(9)-k-k-e 56 3715 3731 530022 e-e-k-d(10)-k-e-k-e 80 3715 3730 530342 e-k-d(10)-k-e-k-e 70 3715 3730 530393 k-d(10)-k-e-k-e-e 46 3716 3732 530023 e-e-k-d(10)-k-e-k-e 74 3716 3731 530090 e-k-k-d(10)-k-k-e 78 3716 3731 530137 e-e-k-d(10)-k-k-e 76 3716 3731 530187 e-d-k-d(10)-k-k-e 68 3716 3731 530237 e-d-d-k-d(9)-k-k-e 36 3716 3731 530287 e-e-e-e-d(9)-k-k-e 56 3716 3731 530343 e-k-d(10)-k-e-k-e 68 3716 3731 530394 k-d(10)-k-e-k-e-e 49 3717 3732 518343 e-e-e-d(10)-k-k-k 5 3717 3733 530024 e-e-k-d(10)-k-e-k-e 79 3717 3732 530091 e-k-k-d(10)-k-k-e 81 3717 3732 530138 e-e-k-d(10)-k-k-e 81 3717 3732 530188 e-d-k-d(10)-k-k-e 78 3717 3732 530238 e-d-d-k-d(9)-k-k-e 29 3717 3732 530288 e-e-e-e-d(9)-k-k-e 69 3717 3732 530344 e-k-d(10)-k-e-k-e 85 3718 3733 530092 e-k-k-d(10)-k-k-e 85 3718 3733 530139 e-e-k-d(10)-k-k-e 79 3718 3733 530189 e-d-k-d(10)-k-k-e 77 3718 3733 530239 e-d-d-k-d(9)-k-k-e 61 3718 3733 530289 e-e-e-e-d(9)-k-k-e 75 3720 3735 528880 e-e-e-d(10)-k-k-k 65 4022 4037 518344 e-e-e-d(10)-k-k-k 89 4234 4249 530395 k-d(10)-k-e-k-e-e 71 4235 4250 528936 e-e-e-d(10)-k-k-k 71 4235 4251 530025 e-e-k-d(10)-k-e-k-e 90 4235 4250 530345 e-k-d(10)-k-e-k-e 93 4235 4250 530396 k-d(10)-k-c-k-c-c 71 4236 4251 528937 e-e-e-d(10)-k-k-k 73 4236 4252 530026 e-e-k-d(10)-k-e-k-e 87 4236 4251 530093 e-k-k-d(10)-k-k-e 95 4236 4251 530140 e-e-k-d(10)-k-k-e 89 4236 4251 530190 e-d-k-d(10)-k-k-e 82 4236 4251 530240 e-d-d-k-d(9)-k-k-e 50 4236 4251 530290 e-e-e-e-d(9)-k-k-e 69 4236 4251 530346 e-k-d(10)-k-e-k-e 89 4237 4252 528938 e-e-e-d(10)-k-k-k 72 4237 4252 530094 e-k-k-d(10)-k-k-e 88 4237 4252 530141 e-e-k-d(10)-k-k-e 80 4237 4252 530191 e-d-k-d(10)-k-k-e 74 4237 4252 530241 e-d-d-k-d(9)-k-k-e 53 4237 4252 530291 e-e-e-e-d(9)-k-k-e 68 4242 4257 528942 e-e-e-d(10)-k-k-k 77 4320 4335 528945 e-e-e-d(10)-k-k-k 74 4439 4454 530096 e-k-k-d(10)-k-k-e 72 4439 4454 530143 e-e-k-d(10)-k-k-e 74 4439 4454 530193 e-d-k-d(10)-k-k-e 62 4439 4454 530243 e-d-d-k-d(9)-k-k-e 34 4439 4454 530293 e-e-e-e-d(9)-k-k-e 59 4488 4504 530063 e-e-k-d(10)-k-e-k-e 74 4488 4503 530382 e-k-d(10)-k-e-k-e 17 4488 4503 530465 e-k-k-d(10)-k-k-e 63 4488 4503 530473 e-e-k-d(10)-k-k-e 45 4488 4503 530481 e-d-k-d(10)-k-k-e 14 4488 4503 530489 e-d-d-k-d(9)-k-k-e 13 4488 4503 530497 e-e-e-e-d(9)-k-k-e 7 4488 4503 530512 k-d(10)-k-e-k-e-e 21 4489 4504 519638 e-k-k-d(10)-k-k-e 86 4489 4504 530177 e-e-k-d(10)-k-k-e 71 4489 4504 530227 e-d-k-d(10)-k-k-e 51 4019 4504 530277 e-d-d-k-d(9)-k-k-e 70 4489 4504 530327 e-e-e-e-d(9)-k-k-e 61 4490 4505 530466 e-k-k-d(10)-k-k-e 82 4490 4505 530474 e-e-k-d(10)-k-k-e 62 4490 4505 530482 e-d-k-d(10)-k-k-e 53 4490 4505 530490 e-d-d-k-d(9)-k-k-e 42 4490 4505 530498 e-e-e-e-d(9)-k-k-e 45 4490 4505 530506 e-k-d(10)-k-e-k-e 70 4539 4554 530433 k-d(10)-k-e-k-e-e 62 4540 4555 528958 e-e-e-d(10)-k-k-k 66 4540 4556 530056 e-e-k-d(10)-k-e-k-e 73 4540 4555 530383 e-k-d(10)-k-e-k-e 64 4541 4556 518345 e-e-e-d(10)-k-k-k 80 4541 4556 519636 e-k-k-d(10)-k-k-e 90 4541 4556 530178 e-e-k-d(10)-k-k-e 86 4541 4556 530228 e-d-k-d(10)-k-k-e 77 4541 4556 530278 e-d-d-k-d(9)-k-k-e 86 4541 4556 530328 e-e-e-e-d(9)-k-k-e 80 4542 4557 528959 e-e-e-d(10)-k-k-k 73 4621 4636 528973 e-e-e-d(10)-k-k-k 71 4782 4797 530329 e-e-e-e-d(9)-k-k-e 61 4813 4829 530032 e-e-k-d(10)-k-e-k-e 74 4813 4828 530099 e-k-k-d(10)-k-k-e 73 4813 4828 530146 e-e-k-d(10)-k-k-e 70 4813 4828 530196 e-d-k-d(10)-k-k-e 67 4813 4828 530246 e-d-d-k-d(9)-k-k-e 39 4813 4828 530296 e-e-e-e-d(9)-k-k-e 67 4813 4828 530352 e-k-d(10)-k-e-k-e 67 4814 4829 530100 e-k-k-d(10)-k-k-e 77 4814 4829 530147 e-e-k-d(10)-k-k-e 84 4814 4829 530197 e-d-k-d(10)-k-k-e 71 4814 4829 530247 e-d-d-k-d(9)-k-k-e 53 4814 4829 530297 e-e-e-e-d(9)-k-k-e 75 4814 4829 530403 k-d(10)-k-e-k-e-e 77 4815 4831 530033 e-e-k-d(10)-k-e-k-e 65 4815 4830 530353 e-k-d(10)-k-e-k-e 83 4816 4831 530101 e-k-k-d(10)-k-k-e 59 4816 4831 530148 e-e-k-d(10)-k-k-e 79 4816 4831 530198 e-d-k-d(10)-k-k-e 54 4816 4831 530248 e-d-d-k-d(9)-k-k-e 32 4816 4831 530298 e-e-e-e-d(9)-k-k-e 73 4827 4842 530404 k-d(10)-k-e-k-e-e 67 4828 4844 530034 e-e-k-d(10)-k-e-k-e 69 4828 4843 530154 e-k-d(10)-k-e-k-e 85 4828 4843 530405 k-d(10)-k-e-k-e-e 55 4829 4845 530035 e-e-k-d(10)-k-e-k-e 69 4829 4844 530102 e-k-k-d(10)-k-k-e 71 4829 4844 530149 e-e-k-d(10)-k-k-e 70 4829 4844 530199 e-d-k-d(10)-k-k-e 58 4829 4844 530249 e-d-d-k-d(9)-k-k-e 47 4829 4844 530299 e-e-e-e-d(9)-k-k-e 47 4829 4844 530355 e-k-d(10)-k-e-k-e 72 4830 4845 530103 e-k-k-d(10)-k-k-e 77 4830 4845 530150 c-c-k-d(10)-k-k-c 73 4830 4845 530200 e-d-k-d(10)-k-k-e 63 4830 4845 530250 e-d-d-k-d(9)-k-k-e 59 4830 4845 530300 e-e-e-e-d(9)-k-k-e 65 4842 4857 530435 k-d(10)-k-e-k-e-e 62 4843 4859 530057 e-e-k-d(10)-k-e-k-e 69 4843 4858 530385 e-k-d(10)-k-e-k-e 70 4844 4859 529005 e-e-e-d(10)-k-k-k 64 4844 4859 530130 e-k-k-d(10)-k-k-e 85 4844 4859 530180 e-e-k-d(10)-k-k-e 82 4844 4859 530230 e-d-k-d(10)-k-k-e 65 4844 4859 530280 e-d-d-k-d(9)-k-k-e 75 4844 4859 530330 e-e-e-e-d(9)-k-k-e 52 e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

TABLE 117 Inhibition of human Target-1 mRNA levels by chimeric antisense oligonucleotides targeted to Target-1 Genomic Sequence Human Human Start Stop % Site Site ISIS No Chemistry inhibition 1794 1809 529022 e-e-e-d(10)-k-k-k 69 1796 1811 529023 e-e-e-d(10)-k-k-k 72 1906 1921 529024 e-e-e-d(10)-k-k-k 64 1907 1922 529025 e-e-e-d(10)-k-k-k 73 1966 1981 529026 e-e-e-d(10)-k-k-k 78 1968 1983 529027 e-e-e-d(10)-k-k-k 92 2409 2425 530038 e-e-k-d(10)-k-e-k-e 71 2409 2424 530358 e-k-d(10)-k-e-k-e 46 2410 2425 530106 e-k-k-d(10)-k-k-e 70 2410 2425 530153 e-e-k-d(10)-k-k-e 50 2410 2425 530203 e-d-k-d(10)-k-k-e 43 2410 2425 530253 e-d-d-k-d(9)-k-k-e 33 2410 2425 530303 e-e-e-e-d(9)-k-k-e 40 2670 2686 530039 e-e-k-d(10)-k-e-k-e 73 2670 2685 530359 e-k-d(10)-k-e-k-e 82 2671 2686 530107 e-k-k-d(10)-k-k-e 77 2671 2686 530154 e-e-k-d(10)-k-k-e 57 2671 2686 530204 e-d-k-d(10)-k-k-e 28 2671 2686 530254 e-d-d-k-d(9)-k-k-e 3 2671 2686 530304 e-e-e-e-d(9)-k-k-e 22 2703 2718 530429 k-d(10)-k-e-k-e-e 60 2704 2720 530065 e-e-k-d(10)-k-e-k-e 70 2704 2719 530379 e-k-d(10)-k-e-k-e 54 2705 2720 530127 e-k-k-d(10)-k-k-e 80 2705 2720 530174 e-e-k-d(10)-k-k-e 69 2705 2720 530224 e-d-k-d(10)-k-k-e 32 2705 2720 530274 e-d-d-k-d(9)-k-k-e 38 2705 2720 530324 e-e-e-e-d(9)-k-k-e 32 5000 5015 530410 k-d(10)-k-e-k-e-e 53 5001 5017 530040 e-e-k-d(10)-k-e-k-e 67 5001 5016 530360 e-k-d(10)-k-e-k-e 70 5002 5017 530108 e-k-k-d(10)-k-k-e 70 5002 5017 530155 e-e-k-d(10)-k-k-e 53 5002 5017 530205 e-d-k-d(10)-k-k-e 44 5002 5017 530255 e-d-d-k-d(9)-k-k-e 33 5002 5017 530305 e-e-e-e-d(9)-k-k-e 22 5699 5714 530411 k-d(10)-k-e-k-e-e 91 5700 5716 530041 e-e-k-d(10)-k-e-k-e 89 5700 5715 530361 e-k-d(10)-k-e-k-e 88 5701 5716 530109 e-k-k-d(10)-k-k-e 89 5701 5716 530156 e-e-k-d(10)-k-k-e 91 5701 5716 530206 e-d-k-d(10)-k-k-e 89 5701 5716 530256 e-d-d-k-d(9)-k-k-e 33 5701 5716 530306 e-e-e-e-d(9)-k-k-e 83 6475 6491 530066 e-e-k-d(10)-k-e-k-e 82 6475 6490 530386 e-k-d(10)-k-e-k-e 53 6476 6491 530131 e-k-k-d(10)-k-k-e 97 6476 6491 530181 e-e-k-d(10)-k-k-e 82 6476 6491 530231 e-d-k-d(10)-k-k-e 75 6476 6491 530281 e-d-d-k-d(9)-k-k-e 69 6476 6491 530331 e-e-e-e-d(9)-k-k-e 53 6846 6861 529039 e-e-e-d(10)-k-k-k 31 8079 8095 530042 e-e-k-d(10)-k-e-k-e 78 8079 8094 530362 e-k-d(10)-k-e-k-e 76 8080 8095 530110 e-k-k-d(10)-k-k-e 84 8080 8095 530157 e-e-k-d(10)-k-k-e 69 8080 8095 530207 e-d-k-d(10)-k-k-e 55 8080 8095 530257 e-d-d-k-d(9)-k-k-e 39 8080 8095 530307 e-e-e-e-d(9)-k-k-e 77 9123 9138 530413 k-d(10)-k-e-k-e-e 73 9862 9877 530414 k-d(10)-k-e-k-e-e 61 9863 9879 530044 e-e-k-d(10)-k-e-k-e 78 9863 9878 530364 e-k-d(10)-k-e-k-e 59 9864 9879 530112 e-k-k-d(10)-k-k-e 84 9864 9879 530159 e-e-k-d(10)-k-k-e 69 9864 9879 530209 e-d-k-d(10)-k-k-e 54 9864 9879 530259 e-d-d-k-d(9)-k-k-e 57 9864 9879 530309 e-e-e-e-d(9)-k-k-e 46 9864 9879 530415 k-d(10)-k-e-k-e-e 51 9865 9881 530045 e-e-k-d(10)-k-e-k-e 73 9865 9880 530365 e-k-d(10)-k-e-k-e 78 9866 9881 530113 e-k-k-d(10)-k-k-e 60 9866 9881 530160 e-e-k-d(10)-k-k-e 54 9866 9881 530210 e-d-k-d(10)-k-k-e 28 9866 9881 530260 e-d-d-k-d(9)-k-k-e 0 9866 9881 530310 e-e-e-e-d(9)-k-k-e 26 9873 9888 530416 k-d(10)-k-e-k-e-e 57 9874 9890 530046 e-e-k-d(10)-k-e-k-e 76 9874 9889 530366 e-k-d(10)-k-e-k-e 75 9874 9889 530417 k-d(10)-k-e-k-e-e 66 9875 9891 530047 e-e-k-d(10)-k-e-k-e 75 9875 9890 530114 e-k-k-d(10)-k-k-e 80 9875 9890 530161 e-e-k-d(10)-k-k-e 81 9875 9890 530211 e-d-k-d(10)-k-k-e 73 9875 9890 530261 e-d-d-k-d(9)-k-k-e 78 9875 9890 530311 e-e-e-e-d(9)-k-k-e 82 9875 9890 530367 e-k-d(10)-k-e-k-e 80 9876 9891 530115 e-k-k-d(10)-k-k-e 74 9876 9891 530162 e-e-k-d(10)-k-k-e 68 9876 9891 530212 e-d-k-d(10)-k-k-e 58 9876 9891 530262 e-d-d-k-d(9)-k-k-e 23 9876 9891 530312 e-e-e-e-d(9)-k-k-e 52 9876 9891 530418 k-d(10)-k-e-k-e-e 59 9877 9893 530048 e-e-k-d(10)-k-e-k-e 82 9877 9892 530368 e-k-d(10)-k-e-k-e 85 9878 9893 530116 e-k-k-d(10)-k-k-e 90 9878 9893 530163 e-e-k-d(10)-k-k-e 79 9878 9893 530213 e-d-k-d(10)-k-k-e 72 9878 9893 530263 e-d-d-k-d(9)-k-k-e 73 9878 9893 530313 e-e-e-e-d(9)-k-k-e 61 12345 12360 530414 k-d(10)-k-e-k-e-e 61 12346 12362 530044 e-e-k-d(10)-k-e-k-e 78 12346 12361 530364 e-k-d(10)-k-e-k-e 59 12347 12362 530112 e-k-k-d(10)-k-k-e 84 12347 12362 530159 e-e-k-d(10)-k-k-e 69 12347 12362 530209 e-d-k-d(10)-k-k-e 54 12347 12362 530259 e-d-d-k-d(9)-k-k-e 57 12347 12362 530309 e-e-e-e-d(9)-k-k-e 46 12347 12362 530415 k-d(10)-k-e-k-e-e 51 12348 12364 530045 e-e-k-d(10)-k-e-k-e 73 12348 12363 530365 e-k-d(10)-k-e-k-e 78 12349 12364 530113 e-k-k-d(10)-k-k-e 60 12349 12364 530160 e-e-k-d(10)-k-k-e 54 12349 12364 530210 e-d-k-d(10)-k-k-e 28 12349 12364 530260 e-d-d-k-d(9)-k-k-e 0 12349 12364 530310 e-e-e-e-d(9)-k-k-e 26 12356 12371 530416 k-d(10)-k-e-k-e-e 57 12357 12373 530046 e-e-k-d(10)-k-e-k-e 76 12357 12372 530366 e-k-d(10)-k-e-k-e 75 12357 12372 530417 k-d(10)-k-e-k-e-e 66 12358 12374 530047 e-e-k-d(10)-k-e-k-e 75 12358 12373 530114 e-k-k-d(10)-k-k-e 80 12358 12373 530161 e-e-k-d(10)-k-k-e 81 12358 12373 530211 e-d-k-d(10)-k-k-e 73 12358 12373 530261 e-d-d-k-d(9)-k-k-e 78 12358 12373 530311 e-e-e-e-d(9)-k-k-e 82 12358 12373 530367 e-k-d(10)-k-e-k-e 80 12359 12374 530115 e-k-k-d(10)-k-k-e 74 12359 12374 530162 e-e-k-d(10)-k-k-e 68 12359 12374 530212 e-d-k-d(10)-k-k-e 58 12359 12374 530262 e-d-d-k-d(9)-k-k-e 23 12359 12374 530312 e-e-e-e-d(9)-k-k-e 52 12359 12374 530418 k-d(10)-k-e-k-e-e 59 12360 12376 530048 e-e-k-d(10)-k-e-k-e 82 12360 12375 530368 e-k-d(10)-k-e-k-e 85 12361 12376 530116 e-k-k-d(10)-k-k-e 90 12361 12376 530163 e-e-k-d(10)-k-k-e 79 12361 12376 530213 e-d-k-d(10)-k-k-e 72 12361 12376 530263 e-d-d-k-d(9)-k-k-e 73 12361 12376 530313 e-e-e-e-d(9)-k-k-e 61 15469 15484 530132 e-k-k-d(10)-k-k-e 74 15469 15484 530182 e-e-k-d(10)-k-k-e 48 15469 15484 530232 e-d-k-d(10)-k-k-e 21 15469 15484 530282 e-d-d-k-d(9)-k-k-e 19 15469 15484 530332 e-e-e-e-d(9)-k-k-e 20 16863 16878 530419 k-d(10)-k-e-k-e-e 75 16864 16880 530049 e-e-k-d(10)-k-e-k-e 88 16864 16879 530369 e-k-d(10)-k-e-k-e 92 16865 16880 530117 e-k-k-d(10)-k-k-e 73 16865 16880 530164 e-e-k-d(10)-k-k-e 65 16865 16880 530214 e-d-k-d(10)-k-k-e 37 16865 16880 530264 e-d-d-k-d(9)-k-k-e 48 16865 16880 530314 e-e-e-e-d(9)-k-k-e 42 25105 25120 530717 e-e-e-d(10)-k-k-k 77 50692 50707 530423 k-d(10)-k-e-k-e-e 70 50693 50709 530053 e-e-k-d(10)-k-e-k-e 84 50693 50708 530373 e-k-d(10)-k-e-k-e 85 50694 50709 530121 e-k-k-d(10)-k-k-e 77 50694 50709 530168 e-e-k-d(10)-k-k-e 75 50694 50709 530218 e-d-k-d(10)-k-k-e 61 50694 50709 530268 e-d-d-k-d(9)-k-k-e 76 50694 50709 530318 e-e-e-e-d(9)-k-k-e 73 51905 51920 528400 e-e-e-d(10)-k-k-k 57 51910 51925 528403 e-e-e-d(10)-k-k-k 72 64959 64974 529082 e-e-e-d(10)-k-k-k 20 66136 66151 530425 k-d(10)-k-e-k-e-e 73 66137 66153 530054 e-e-k-d(10)-k-e-k-e 75 66137 66152 530375 e-k-d(10)-k-e-k-e 77 66138 66153 530123 e-k-k-d(10)-k-k-e 86 66138 66153 530170 e-e-k-d(10)-k-k-e 87 66138 66153 530220 e-d-k-d(10)-k-k-e 74 66138 66153 530270 e-d-d-k-d(9)-k-k-e 87 66138 66153 530320 e-e-e-e-d(9)-k-k-e 83 66184 66200 530059 e-e-k-d(10)-k-e-k-e 73 66184 66199 530376 e-k-d(10)-k-e-k-e 77 66185 66200 530124 e-k-k-d(10)-k-k-e 79 66185 66200 530171 e-e-k-d(10)-k-k-e 69 66185 66200 530221 e-d-k-d(10)-k-k-e 64 66185 66200 530271 e-d-d-k-d(9)-k-k-e 73 66185 66200 530321 e-e-e-e-d(9)-k-k-e 56 67067 67083 530060 e-e-k-d(10)-k-e-k-e 77 67067 67082 530377 e-k-d(10)-k-e-k-e 66 67068 67083 530125 e-k-k-d(10)-k-k-e 65 67068 67083 530172 e-e-k-d(10)-k-k-e 59 67068 67083 530222 e-d-k-d(10)-k-k-e 48 67068 67083 530272 e-d-d-k-d(9)-k-k-e 63 67068 67083 530322 e-e-e-e-d(9)-k-k-e 45 71616 71631 530120 e-k-k-d(10)-k-k-e 78 71616 71631 530167 e-e-k-d(10)-k-k-e 69 71616 71631 530217 e-d-k-d(10)-k-k-e 47 71616 71631 530267 e-d-d-k-d(9)-k-k-e 64 71616 71631 530317 e-e-e-e-d(9)-k-k-e 60 73868 73883 530126 e-k-k-d(10)-k-k-e 70 73868 73883 530173 e-e-k-d(10)-k-k-e 62 73868 73883 530223 e-d-k-d(10)-k-k-e 44 73868 73883 530273 e-d-d-k-d(9)-k-k-e 63 73868 73883 530323 e-e-e-e-d(9)-k-k-e 37 74199 74214 530513 k-d(10)-k-e-k-e-e 88 74200 74215 530507 e-k-d(10)-k-e-k-e 86 74200 74215 530514 k-d(10)-k-e-k-e-e 80 74201 74216 530430 k-d(10)-k-e-k-e-e 87 74201 74216 530468 e-k-k-d(10)-k-k-e 81 74201 74216 530476 e-e-k-d(10)-k-k-e 82 74201 74216 530484 e-d-k-d(10)-k-k-e 74 74201 74216 530492 e-d-d-k-d(9)-k-k-e 83 74201 74216 530500 e-e-e-e-d(9)-k-k-e 56 74201 74216 530508 e-k-d(10)-k-e-k-e 83 74202 74218 530062 e-e-k-d(10)-k-e-k-e 94 74202 74217 530380 e-k-d(10)-k-e-k-e 94 74202 74217 530469 e-k-k-d(10)-k-k-e 91 74202 74217 530477 e-e-k-d(10)-k-k-e 87 74202 74217 530485 e-d-k-d(10)-k-k-e 87 74202 74217 530493 e-d-d-k-d(9)-k-k-e 81 74202 74217 530501 e-e-e-e-d(9)-k-k-e 74 74202 74217 530515 k-d(10)-k-e-k-e-e 87 74203 74218 481464 k-k-k-d(10)-k-k-k 93 74203 74218 518349 e-e-e-d(10)-k-k-k 58 74203 74218 519637 e-k-k-d(10)-k-k-e 96 74203 74218 530175 e-e-k-d(10)-k-k-e 93 74203 74218 530225 e-d-k-d(10)-k-k-e 85 74203 74218 530275 e-d-d-k-d(9)-k-k-e 91 74203 74218 530325 e-e-e-e-d(9)-k-k-e 91 74204 74219 530470 e-k-k-d(10)-k-k-e 91 74204 74219 530478 e-e-k-d(10)-k-k-e 87 74204 74219 530486 e-d-k-d(10)-k-k-e 84 74204 74219 530494 e-d-d-k-d(9)-k-k-e 60 74204 74219 530502 e-e-e-e-d(9)-k-k-e 64 74204 74219 530509 e-k-d(10)-k-e-k-e 80 74205 74220 530471 e-k-k-d(10)-k-k-e 83 74205 74220 530479 e-e-k-d(10)-k-k-e 74 74205 74220 530487 e-d-k-d(10)-k-k-e 71 74205 74220 530495 e-d-d-k-d(9)-k-k-e 68 74205 74220 530503 e-e-e-e-d(9)-k-k-e 53 74648 74663 530128 e-k-k-d(10)-k-k-e 52 74648 74663 530176 e-e-k-d(10)-k-k-e 66 74648 74663 530226 e-d-k-d(10)-k-k-e 51 74648 74663 530276 e-d-d-k-d(9)-k-k-e 70 74648 74663 530326 e-e-e-e-d(9)-k-k-e 52 74734 74749 528866 e-e-e-d(10)-k-k-k 60 74735 74750 528867 e-e-e-d(10)-k-k-k 47 74772 74787 530086 e-k-k-d(10)-k-k-e 58 74772 74787 530133 e-e-k-d(10)-k-k-e 53 74772 74787 530183 e-d-k-d(10)-k-k-e 52 74772 74787 530233 e-d-d-k-d(9)-k-k-e 29 74772 74787 530283 e-e-e-e-d(9)-k-k-e 32 74782 74797 530390 k-d(10)-k-e-k-e-e 83 74783 74798 530340 e-k-d(10)-k-e-k-e 89 74784 74799 528869 e-e-e-d(10)-k-k-k 83 74784 74799 530088 e-k-k-d(10)-k-k-e 90 74784 74799 530135 e-e-k-d(10)-k-k-e 91 74784 74799 530185 e-d-k-d(10)-k-k-e 85 74784 74799 530235 e-d-d-k-d(9)-k-k-e 28 74784 74799 530285 e-e-e-e-d(9)-k-k-e 86 74784 74799 530391 k-d(10)-k-e-k-e-e 79 74785 74801 530021 e-e-k-d(10)-k-e-k-e 87 74785 74800 530341 e-k-d(10)-k-e-k-e 88 74786 74801 530089 e-k-k-d(10)-k-k-e 71 74786 74801 530136 e-e-k-d(10)-k-k-e 66 74786 74801 530186 e-d-k-d(10)-k-k-e 51 74786 74801 530236 e-d-d-k-d(9)-k-k-e 74 74786 74801 530286 e-e-e-e-d(9)-k-k-e 56 74902 74918 530022 e-e-k-d(10)-k-e-k-e 80 74902 74917 530342 e-k-d(10)-k-e-k-e 70 74902 74917 530393 k-d(10)-k-e-k-e-e 46 74903 74919 530023 e-e-k-d(10)-k-e-k-e 74 74903 74918 530090 e-k-k-d(10)-k-k-e 78 74903 74918 530137 e-e-k-d(10)-k-k-e 76 74903 74918 530187 e-d-k-d(10)-k-k-e 68 74903 74918 530237 e-d-d-k-d(9)-k-k-e 36 74903 74918 530287 e-e-e-e-d(9)-k-k-e 56 74903 74918 530343 e-k-d(10)-k-e-k-e 68 74903 74918 530394 k-d(10)-k-e-k-e-e 49 74904 74919 518343 e-e-e-d(10)-k-k-k 5 74904 74920 530024 e-e-k-d(10)-k-e-k-e 79 74904 74919 530091 e-k-k-d(10)-k-k-e 81 74904 74919 530138 e-e-k-d(10)-k-k-e 81 74904 74919 530188 e-d-k-d(10)-k-k-e 78 74904 74919 530238 e-d-d-k-d(9)-k-k-e 29 74904 74919 530288 e-e-e-e-d(9)-k-k-e 69 74904 74919 530344 e-k-d(10)-k-e-k-e 85 74905 74920 530092 c-k-k-d(10)-k-k-e 85 74905 74920 530139 e-e-k-d(10)-k-k-e 79 74905 74920 530189 e-d-k-d(10)-k-k-e 77 74905 74920 530239 e-d-d-k-d(9)-k-k-e 61 74905 74920 530289 e-e-e-e-d(9)-k-k-e 75 74907 74922 528880 e-e-e-d(10)-k-k-k 65 75209 75224 518344 e-e-e-d(10)-k-k-k 89 75421 75436 530395 k-d(10)-k-e-k-e-e 71 75422 75437 528936 e-e-e-d(10)-k-k-k 71 75422 75438 530025 e-e-k-d(10)-k-e-k-e 90 75422 75437 530345 e-k-d(10)-k-e-k-e 93 75422 75437 530396 k-d(10)-k-e-k-e-e 71 75423 75438 528937 e-e-e-d(10)-k-k-k 73 75423 75439 530026 e-e-k-d(10)-k-e-k-e 87 75423 75438 530093 e-k-k-d(10)-k-k-e 95 75423 75438 530140 e-e-k-d(10)-k-k-e 89 75423 75438 530190 e-d-k-d(10)-k-k-e 82 75423 75438 530240 e-d-d-k-d(9)-k-k-e 50 75423 75438 530290 e-e-e-e-d(9)-k-k-e 69 75423 75438 530346 e-k-d(10)-k-e-k-e 89 75424 75439 528938 e-e-e-d(10)-k-k-k 72 75424 75439 530094 e-k-k-d(10)-k-k-e 88 75424 75439 530141 e-e-k-d(10)-k-k-e 80 75424 75439 530191 e-d-k-d(10)-k-k-e 74 75424 75439 530241 e-d-d-k-d(9)-k-k-e 53 75424 75439 530291 e-e-e-e-d(9)-k-k-e 68 75429 75444 528942 e-e-e-d(10)-k-k-k 77 75492 75507 528944 e-e-e-d(10)-k-k-k 28 75507 75522 528945 e-e-e-d(10)-k-k-k 74 75626 75641 530096 e-k-k-d(10)-k-k-e 72 75626 75641 530143 e-e-k-d(10)-k-k-e 74 75626 75641 530193 e-d-k-d(10)-k-k-e 62 75626 75641 530243 e-d-d-k-d(9)-k-k-e 34 75626 75641 530293 e-e-e-e-d(9)-k-k-e 59 75676 75691 519638 e-k-k-d(10)-k-k-e 86 75676 75691 530177 e-e-k-d(10)-k-k-e 71 75676 75691 530227 e-d-k-d(10)-k-k-e 51 75676 75691 530277 e-d-d-k-d(9)-k-k-e 70 75676 75691 530327 e-e-e-e-d(9)-k-k-e 61 75677 75692 530466 e-k-k-d(10)-k-k-e 82 75677 75692 530474 e-e-k-d(10)-k-k-e 62 75677 75692 530482 e-d-k-d(10)-k-k-e 53 75677 75692 530490 e-d-d-k-d(9)-k-k-e 42 75677 75692 530498 e-e-e-e-d(9)-k-k-e 45 75677 75692 530506 e-k-d(10)-k-e-k-e 70 75726 75741 530433 k-d(10)-k-e-k-e-e 62 75727 75742 528958 e-e-e-d(10)-k-k-k 66 75727 75743 530056 e-e-k-d(10)-k-e-k-e 73 75727 75742 530383 e-k-d(10)-k-e-k-e 64 75728 75743 518345 e-e-e-d(10)-k-k-k 80 75728 75743 519636 e-k-k-d(10)-k-k-e 90 75728 75743 530178 e-e-k-d(10)-k-k-e 86 75728 75743 530228 e-d-k-d(10)-k-k-e 77 75728 75743 530278 e-d-d-k-d(9)-k-k-e 86 75728 75743 530328 e-e-e-e-d(9)-k-k-e 80 75729 75744 528959 e-e-e-d(10)-k-k-k 73 75808 75823 528973 e-e-e-d(10)-k-k-k 71 75969 75984 528995 e-e-e-d(10)-k-k-k 64 75969 75984 530129 e-k-k-d(10)-k-k-e 79 75969 75984 530179 e-e-k-d(10)-k-k-e 74 75969 75984 530229 e-d-k-d(10)-k-k-e 64 75969 75984 530279 e-d-d-k-d(9)-k-k-e 55 75969 75984 530329 e-e-e-e-d(9)-k-k-e 61 75999 76014 530402 k-d(10)-k-e-k-e-e 60 76000 76016 530032 e-e-k-d(10)-k-e-k-e 74 76000 76015 530099 e-k-k-d(10)-k-k-e 73 76000 76015 530146 e-e-k-d(10)-k-k-e 70 76000 76015 530196 e-d-k-d(10)-k-k-e 67 76000 76015 530246 e-d-d-k-d(9)-k-k-e 39 76000 76015 530296 e-e-e-e-d(9)-k-k-e 67 76000 76015 530352 e-k-d(10)-k-e-k-e 67 76001 76016 530100 e-k-k-d(10)-k-k-e 77 76001 76016 530147 e-e-k-d(10)-k-k-e 84 76001 76016 530197 e-d-k-d(10)-k-k-e 71 76001 76016 530247 e-d-d-k-d(9)-k-k-e 53 76001 76016 530297 e-e-e-e-d(9)-k-k-e 75 76001 76016 530403 k-d(10)-k-e-k-e-e 77 76002 76018 530033 e-e-k-d(10)-k-e-k-e 65 76002 76017 530353 e-k-d(10)-k-e-k-e 83 76003 76018 530101 e-k-k-d(10)-k-k-e 59 76003 76018 530148 e-e-k-d(10)-k-k-e 79 76003 76018 530198 e-d-k-d(10)-k-k-e 54 76003 76018 530248 e-d-d-k-d(9)-k-k-e 32 76003 76018 530298 e-e-e-e-d(9)-k-k-e 73 76014 76029 530404 k-d(10)-k-e-k-e-e 67 76015 76031 530034 e-e-k-d(10)-k-e-k-e 69 76015 76030 530354 e-k-d(10)-k-e-k-e 85 76015 76030 530405 k-d(10)-k-e-k-e-e 55 76016 76032 530035 e-e-k-d(10)-k-e-k-e 69 76016 76031 530102 e-k-k-d(10)-k-k-e 71 76016 76031 530149 e-e-k-d(10)-k-k-e 70 76016 76031 530199 e-d-k-d(10)-k-k-e 58 76016 76031 530249 e-d-d-k-d(9)-k-k-e 47 76016 76031 530299 e-e-e-e-d(9)-k-k-e 47 76016 76031 530355 e-k-d(10)-k-e-k-e 72 76017 76032 530103 e-k-k-d(10)-k-k-e 77 76017 76032 530150 e-e-k-d(10)-k-k-e 73 76017 76032 530200 e-d-k-d(10)-k-k-e 63 76017 76032 530250 e-d-d-k-d(9)-k-k-e 59 76017 76032 530300 e-e-e-e-d(9)-k-k-e 65 76029 76044 530435 k-d(10)-k-e-k-e-e 62 76030 76046 530057 e-e-k-d(10)-k-e-k-e 69 76030 76045 530385 e-k-d(10)-k-e-k-e 70 76031 76046 529005 e-e-e-d(10)-k-k-k 64 76031 76046 530130 e-k-k-d(10)-k-k-e 85 76031 76046 530180 e-e-k-d(10)-k-k-e 82 76031 76046 530230 e-d-k-d(10)-k-k-e 65 76031 76046 530280 e-d-d-k-d(9)-k-k-e 75 76031 76046 530330 e-e-e-e-d(9)-k-k-e 52 e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

Example 71 Dose-Dependent Antisense Inhibition of Human Target-1 in HuVEC Cells

Gapmers from the study described in Example 70 exhibiting in vitro inhibition of Target-1 were tested at various doses in HuVEC cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 39.1 nM, 156.3 nM, 625.0 nM, and 2,500.0 nM concentrations of antisense oligonucleotide, as specified in Table 118. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-1 mRNA levels were measured by quantitative real-time PCR. Target-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-1, relative to untreated control cells.

TABLE 118 Dose-dependent antisense inhibition of human Target-1 in HuVEC cells 39.1 625.0 2500.0 IC₅₀ ISIS No nM 156.3 nM nM nM (uM) 481464 6 51 84 94 0.2 518345 0 9 56 84 0.6 518349 16 3 47 83 0.6 519636 16 41 75 89 0.2 519637 24 43 84 94 0.2 519638 6 34 70 92 0.3 528403 0 4 39 77 0.9 528458 0 15 46 81 0.7 528475 1 10 51 76 0.7 528476 0 11 42 80 0.7 528869 25 19 67 86 0.3 528880 0 3 45 76 0.8 528937 0 1 49 82 0.8 528938 0 9 50 82 0.7 528942 0 20 59 88 0.5 528959 0 4 55 79 0.7 529022 0 0 52 81 0.8 529023 0 0 53 90 0.6 529024 0 0 47 80 0.8 529025 0 11 50 90 0.6 529026 0 31 73 96 0.4 529027 0 7 36 80 0.9 530021 6 30 69 92 0.3 530025 10 33 73 92 0.3 530026 3 18 52 80 0.6 530041 0 28 72 91 0.4 530048 0 22 53 83 0.5 530049 2 16 69 92 0.4 530053 0 16 66 90 0.5 530062 4 56 85 94 0.2 530066 0 12 46 84 0.7 530088 2 39 77 93 0.3 530091 3 12 59 84 0.5 530092 7 27 65 85 0.4 530093 7 46 79 96 0.2 530094 0 17 63 89 0.5 530109 9 30 72 94 0.3 530110 0 23 61 83 0.5 530112 0 13 42 90 0.6 530114 0 21 62 79 0.6 530116 22 40 71 92 0.2 530123 8 19 72 93 0.3 530130 0 33 64 89 0.4 530131 4 34 81 93 0.3 530135 22 38 79 94 0.2 530138 6 23 57 86 0.4 530140 4 22 62 91 0.4 530147 0 15 51 83 0.6 530156 7 41 81 96 0.2 530161 0 20 46 78 0.7 530170 0 29 67 90 0.4 530175 37 52 84 95 0.1 530178 8 24 70 86 0.4 530180 0 0 61 82 0.6 530181 0 27 52 86 0.5 530185 0 22 54 86 0.5 530190 17 17 60 87 0.4 530206 8 29 73 93 0.3 530225 0 27 67 91 0.4 530228 11 16 64 86 0.4 530261 5 25 57 91 0.4 530270 7 11 62 91 0.4 530275 14 34 73 91 0.3 530278 1 27 60 85 0.4 530285 5 20 61 82 0.5 530306 3 14 66 85 0.5 530311 6 27 59 86 0.4 530320 3 17 56 85 0.5 530325 5 35 70 92 0.3 530328 4 34 61 87 0.4 530340 8 34 74 90 0.3 530341 2 23 77 89 0.4 530344 16 20 64 89 0.4 530345 15 35 77 94 0.2 530346 5 24 66 92 0.4 530353 7 25 57 83 0.5 530354 2 24 60 81 0.5 530359 0 4 44 89 0.7 530361 13 30 59 92 0.3 530365 0 0 45 88 0.7 530367 0 15 49 88 0.5 530368 0 27 64 89 0.4 530369 10 28 78 95 0.3 530373 13 29 64 92 0.3 530375 0 14 53 90 0.5 530380 8 40 80 94 0.2 530390 11 21 66 90 0.4 530391 20 7 49 86 0.5 530411 5 19 81 95 0.3 530430 0 8 53 91 0.6 530466 0 4 53 87 0.6 530468 4 17 65 90 0.4 530469 8 38 86 94 0.2 530470 5 39 78 91 0.3 530471 0 21 69 91 0.4 530476 7 9 32 89 0.7 530477 0 12 64 87 0.5 530478 0 14 59 90 0.5 530485 0 10 61 85 0.5 530486 0 17 64 80 0.5 530492 0 25 71 89 0.4 530493 4 23 58 88 0.4 530507 5 17 65 82 0.5 530508 0 14 56 89 0.5 530509 0 17 54 86 0.5 530513 6 24 74 91 0.3 530514 1 7 52 78 0.7 530515 0 19 73 89 0.4

Example 72 Antisense Inhibition of Human Target-1 in HuVEC Cells

Additional antisense oligonucleotides were designed targeting a Target-1 nucleic acid and were tested for their effects on Target-1 mRNA in vitro. Cultured HuVEC cells at a density of 20,000 cells per well were transfected using electroporation with 1,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-1 mRNA levels were measured by quantitative real-time PCR. Target-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-1, relative to untreated control cells.

The chemistry column of Table 119 presents the sugar motif of each gapmer, where ‘e’ indicates a 2′-MOE nucleoside, ‘k’ indicates a constrained ethyl (cEt) nucleoside, and ‘d’ indicates a 2′-deoxynucleoside. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5′-methylcytosines.

“Human Target start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Human Target stop site” indicates the 3′-most nucleoside to which the gapmer is targeted in the human gene sequence. Each gapmer listed in Table 119 is targeted to human Target-1 mRNA. Each gapmer listed in Table 120 is targeted to human Target-1 genomic sequence, truncated from nucleotides 4185000 to 4264000).

TABLE 119 Inhibition of human Target-1 mRNA levels by chimeric antisense oligonucleotides targeted to Target-1 mRNA Human Human % Start Site Stop Site ISIS No Chemistry inhibition 730 745 530011 k-k-k-d(10)-e-e-e 73 1901 1916 529974 e-e-e-d(10)-k-k-k 83 1901 1916 530012 k-k-k-d(10)-e-e-e 73 2206 2221 530015 k-k-k-d(10)-e-e-e 38 3016 3031 481464 k-k-k-d(10)-k-k-k 94 3461 3476 529975 e-e-e-d(10)-k-k-k 54 3461 3476 530013 k-k-k-d(10)-e-e-e 58 3584 3600 530018 e-e-k-d(10)-k-e-k-e 46 3585 3600 529944 e-e-e-d(10)-k-k-k 44 3585 3600 529977 k-k-k-d(10)-e-e-e 66 3592 3608 530019 e-e-k-d(10)-k-e-k-e 43 3593 3608 529945 e-e-e-d(10)-k-k-k 22 3593 3608 529978 k-k-k-d(10)-e-e-e 49 3596 3612 530020 e-e-k-d(10)-k-e-k-e 85 3597 3612 529979 k-k-k-d(10)-e-e-e 86 3599 3614 529946 e-e-e-d(10)-k-k-k 46 3599 3614 529980 k-k-k-d(10)-e-e-e 25 3716 3731 529947 e-e-e-d(10)-k-k-k 68 3716 3731 529981 k-k-k-d(10)-e-e-e 83 3718 3733 529948 e-e-e-d(10)-k-k-k 75 3718 3733 529982 k-k-k-d(10)-e-e-e 84 4236 4251 529983 k-k-k-d(10)-e-e-e 96 4237 4252 529984 k-k-k-d(10)-e-e-e 91 4437 4452 529949 e-e-e-d(10)-k-k-k 48 4437 4452 529985 k-k-k-d(10)-e-e-e 37 4439 4454 529950 e-e-e-d(10)-k-k-k 58 4439 4454 529986 k-k-k-d(10)-e-e-e 72 4646 4661 529987 k-k-k-d(10)-e-e-e 0 4664 4679 529951 e-e-e-d(10)-k-k-k 38 4664 4679 529988 k-k-k-d(10)-e-e-e 40 4782 4797 530016 k-k-k-d(10)-e-e-e 60 4813 4828 529952 e-e-e-d(10)-k-k-k 65 4813 4828 529989 k-k-k-d(10)-e-e-e 63 4814 4829 529953 e-e-e-d(10)-k-k-k 65 4814 4829 529990 k-k-k-d(10)-e-e-e 75 4816 4831 529954 e-e-e-d(10)-k-k-k 79 4816 4831 529991 k-k-k-d(10)-e-e-e 52 4829 4844 529955 e-e-e-d(10)-k-k-k 52 4829 4844 529992 k-k-k-d(10)-e-e-e 23 4830 4845 529956 e-e-e-d(10)-k-k-k 60 4830 4845 529993 k-k-k-d(10)-e-e-e 51 4844 4859 530014 k-k-k-d(10)-e-e-e 67 e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

TABLE 120 Inhibition of human TARGET-1 mRNA levels by chimeric antisense oligonucleotides targeted to Target-1 Genomic Sequence Human Human % Start Site Stop Site Sequence Chemistry inhibition 74203 74218 481464 k-k-k-d(10)-k-k-k 94 74772 74787 529944 e-e-e-d(10)-k-k-k 44 74780 74795 529945 e-e-e-d(10)-k-k-k 22 74786 74801 529946 e-e-e-d(10)-k-k-k 46 74903 74918 529947 e-e-e-d(10)-k-k-k 68 74905 74920 529948 e-e-e-d(10)-k-k-k 75 75624 75639 529949 e-e-e-d(10)-k-k-k 48 75626 75641 529950 e-e-e-d(10)-k-k-k 58 75851 75866 529951 e-e-e-d(10)-k-k-k 38 76000 76015 529952 e-e-e-d(10)-k-k-k 65 76001 76016 529953 e-e-e-d(10)-k-k-k 65 76003 76018 529954 e-e-e-d(10)-k-k-k 79 76016 76031 529955 e-e-e-d(10)-k-k-k 52 76017 76032 529956 e-e-e-d(10)-k-k-k 60 2340 2355 529957 e-e-e-d(10)-k-k-k 21 2385 2400 529958 e-e-e-d(10)-k-k-k 10 2410 2425 529959 e-e-e-d(10)-k-k-k 51 2671 2686 529960 e-e-e-d(10)-k-k-k 30 5002 5017 529961 e-e-e-d(10)-k-k-k 52 5701 5716 529962 e-e-e-d(10)-k-k-k 91 8080 8095 529963 e-e-e-d(10)-k-k-k 55 9125 9140 529964 e-e-e-d(10)-k-k-k 18 11263 11278 529964 e-e-e-d(10)-k-k-k 18 9864 9879 529965 e-e-e-d(10)-k-k-k 52 12347 12362 529965 e-e-e-d(10)-k-k-k 52 9866 9881 529966 e-e-e-d(10)-k-k-k 51 12349 12364 529966 e-e-e-d(10)-k-k-k 51 9875 9890 529967 e-e-e-d(10)-k-k-k 80 12358 12373 529967 e-e-e-d(10)-k-k-k 80 9876 9891 529968 e-e-e-d(10)-k-k-k 56 12359 12374 529968 e-e-e-d(10)-k-k-k 56 9878 9893 529969 e-e-e-d(10)-k-k-k 69 12361 12376 529969 e-e-e-d(10)-k-k-k 69 16865 16880 529970 e-e-e-d(10)-k-k-k 41 26063 26078 529971 e-e-e-d(10)-k-k-k 32 48404 48419 529972 e-e-e-d(10)-k-k-k 30 71616 71631 529973 e-e-e-d(10)-k-k-k 49 66138 66153 529974 e-e-e-d(10)-k-k-k 83 74648 74663 529975 e-e-e-d(10)-k-k-k 54 2705 2720 529976 e-e-e-d(10)-k-k-k 25 74772 74787 529977 k-k-k-d(10)-e-e-e 66 74780 74795 529978 k-k-k-d(10)-e-e-e 49 74784 74799 529979 k-k-k-d(10)-e-e-e 86 74786 74801 529980 k-k-k-d(10)-e-e-e 25 74903 74918 529981 k-k-k-d(10)-e-e-e 83 74905 74920 529982 k-k-k-d(10)-e-e-e 84 75423 75438 529983 k-k-k-d(10)-e-e-e 96 75424 75439 529984 k-k-k-d(10)-e-e-e 91 75624 75639 529985 k-k-k-d(10)-e-e-e 37 75626 75641 529986 k-k-k-d(10)-e-e-e 72 75833 75848 529987 k-k-k-d(10)-e-e-e 0 75851 75866 529988 k-k-k-d(10)-e-e-e 40 76000 76015 529989 k-k-k-d(10)-e-e-e 63 76001 76016 529990 k-k-k-d(10)-e-e-e 75 76003 76018 529991 k-k-k-d(10)-e-e-e 52 76016 76031 529992 k-k-k-d(10)-e-e-e 23 76017 76032 529993 k-k-k-d(10)-e-e-e 51 2340 2355 529994 k-k-k-d(10)-e-e-e 44 2385 2400 529995 k-k-k-d(10)-e-e-e 0 2410 2425 529996 k-k-k-d(10)-e-e-e 65 2671 2686 529997 k-k-k-d(10)-e-e-e 44 5002 5017 529998 k-k-k-d(10)-e-e-e 35 5701 5716 529999 k-k-k-d(10)-e-e-e 91 8080 8095 530000 k-k-k-d(10)-e-e-e 80 9125 9140 530001 k-k-k-d(10)-e-e-e 21 11263 11278 530001 k-k-k-d(10)-e-e-e 21 9864 9879 530002 k-k-k-d(10)-e-e-e 74 12347 12362 530002 k-k-k-d(10)-e-e-e 74 9866 9881 530003 k-k-k-d(10)-e-e-e 67 12349 12364 530003 k-k-k-d(10)-e-e-e 67 9875 9890 530004 k-k-k-d(10)-e-e-e 83 12358 12373 530004 k-k-k-d(10)-e-e-e 83 9876 9891 530005 k-k-k-d(10)-e-e-e 77 12359 12374 530005 k-k-k-d(10)-e-e-e 77 9878 9893 530006 k-k-k-d(10)-e-e-e 89 12361 12376 530006 k-k-k-d(10)-e-e-e 89 16865 16880 530007 k-k-k-d(10)-e-e-e 21 26063 26078 530008 k-k-k-d(10)-e-e-e 58 48404 48419 530009 k-k-k-d(10)-e-e-e 59 71616 71631 530010 k-k-k-d(10)-e-e-e 75 50694 50709 530011 k-k-k-d(10)-e-e-e 73 66138 66153 530012 k-k-k-d(10)-e-e-e 73 74648 74663 530013 k-k-k-d(10)-e-e-e 58 76031 76046 530014 k-k-k-d(10)-e-e-e 67 67068 67083 530015 k-k-k-d(10)-e-e-e 38 75969 75984 530016 k-k-k-d(10)-e-e-e 60 2705 2720 530017 k-k-k-d(10)-e-e-e 46 74771 74787 530018 e-e-k-d(10)-k-e-k-e 46 74779 74795 530019 e-e-k-d(10)-k-e-k-e 43 74783 74799 530020 e-e-k-d(10)-k-e-k-e 85 e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside

Example 73 Dose-Dependent Antisense Inhibition of Human Target-1 in HuVEC Cells

Gapmers from the study described in Example 72 exhibiting in vitro inhibition of Target-1 were tested at various doses in HuVEC cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 39.1 nM, 156.3 nM, 625.0 nM, and 2,500.0 nM concentrations of antisense oligonucleotide, as specified in Table 121. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-1 mRNA levels were measured by quantitative real-time PCR. Target-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-1, relative to untreated control cells.

TABLE 121 Dose-dependent antisense inhibition of human Target-1 in HuVEC cells 39.1 156.3 625.0 2500.0 IC₅₀ ISIS No nM nM nM nM (μM) 481464 41 78 92 91 0.04 529962 30 51 86 95 0.12 529979 0 43 81 95 0.27 529982 0 0 70 90 0.56 529983 31 67 87 94 0.08 529984 17 44 83 97 0.19 529999 29 51 83 96 0.13 530006 18 38 77 94 0.22 530020 2 39 75 92 0.28 

We claim: 1.-238. (canceled)
 239. A compound comprising: a modified oligonucleotide consisting of 10 to 20 linked nucleosides, wherein the modified oligonucleotide comprises: a 5′-wing consisting of 2 to 5 linked nucleosides; a 3′-wing consisting of 2 to 5 linked nucleosides; and a gap between the 5′-wing and the 3′-wing consisting of 6 to 14 linked 2′-deoxynucleosides; and wherein at least one of the 5′-wing and the 3′-wing comprises at least one bicyclic nucleoside selected from among LNA, α-L-LNA, or ENA; at least one of the 5′-wing and the 3′-wing comprises at least one 2′-substituted nucleoside; and at least one of the 5′-wing and the 3′-wing comprises at least one 2′-deoxynucleoside; and wherein the nucleobase sequence of the modified oligonucleotide is complementary to the nucleobase sequence of a target nucleic acid.
 240. The compound of claim 239, wherein each of the 5′-wing and the 3′-wing comprises at least one 2′-deoxynucleoside.
 241. The compound of claim 239, wherein the 3′-wing comprises at least one 2′-deoxynucleoside.
 242. The compound of claim 239, wherein the 5′-wing comprises at least one 2′-deoxynucleoside.
 243. The compound of claim 239, wherein the 2′-substituent of the at least one 2′-substituted nucleoside is selected from among OCH₃, F, OCH₂F, OCHF₂, OCF₃, OCH₂CH₃, O(CH₂)₂F, OCH₂CHF₂, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—SCH₃, O(CH₂)₂—OCF₃, O(CH₂)₃—N(R₄)(R₅), O(CH₂)₂—ON(R₄)(R₅), O(CH₂)₂—O(CH₂)₂—N(R₄)(R₅), OCH₂C(═O)—N(R₄)(R₅), OCH₂C(═O)—N(R₆)—(CH₂)₂—N(R₄)(R₅) and O(CH₂)₂—N(R₆)—C(═NR₇)[N(R₄)(R₅)] wherein R₄, R₅, R₆ and R₇ are each, independently, H or C₁-C₆ alkyl.
 244. The compound of claim 239, wherein the 2′-substituent of the at least one 2′-substituted nucleoside is selected from among OCH₃, F, and O(CH₂)₂—OCH₃.
 245. The compound of claim 244, wherein the 2′-substituent of the at least one 2′-substituted nucleoside is O(CH₂)₂—OCH₃.
 246. The compound of claim 244, wherein the 2′-substituent of the at least one 2′-substituted nucleoside is OCH₃.
 247. The compound of claim 239, wherein the 5′-wing does not comprise a bicyclic nucleotide.
 248. The compound of claim 239, wherein the 3′-wing does not comprise a bicyclic nucleotide.
 249. The compound of claim 239, wherein the 5′-wing consists of 2 linked nucleosides.
 250. The compound of claim 239, wherein the 5′-wing consists of 3 linked nucleosides.
 251. The compound of claim 239, wherein the 5′-wing consists of 4 linked nucleosides.
 252. The compound of claim 239, wherein the 5′-wing consists of 5 linked nucleosides.
 253. The compound of claim 239, wherein the gap consists of 9 linked 2′-deoxynucleosides.
 254. The compound of claim 239, wherein the gap consists of 10 linked 2′-deoxynucleosides.
 255. The compound of claim 239, wherein the oligonucleotide consists of 18 linked nucleosides.
 256. The compound of claim 239, wherein the oligonucleotide consists of 20 linked nucleosides.
 257. The compound of claim 239, wherein each internucleoside linkage is a modified internucleoside linkage.
 258. The compound of claim 239, wherein the modified internucleoside linkage is a phosphorothioate linkage. 